GFP antibody

• Catalog Number: orb345286
• Category: Antibodies
• Description: GFP antibody
• Species/Host: Goat
• Clonality: Polyclonal
• Tested applications: ELISA, FC, IF, IHC, IP, WB
• Reactivity: Other
• Isotype: IgG
• Immunogen: The immunogen is a Green Fluorescent Protein (GFP) fusion protein corresponding to the full length amino acid sequence (246aa) derived from the jellyfish Aequorea victoria.
• Concentration: 1.1 mg/mL
• Dilution range: ELISA: 1:10,000 - 1:30,000, FC: User Optimized, IHC: 1:200 - 1:1,000, IF: 1:500, IP: User Optimized, WB: 1:1,000 - 1:10,000
• Form/Appearance: Liquid (sterile filtered)
• Purity: GFP antibody was prepared from monospecific antiserum by immunoaffinity chromatography using Green Fluorescent Protein (Aequorea victoria) coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Goat Serum and purified and partially purified Green Fluorescent Protein (Aequorea victoria). No reaction was observed against Human, Mouse or Rat serum proteins.
• Conjugation: Unconjugated
• UniProt ID: P42212
• Storage: Store GFP antibody at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Alternative names: goat anti-GFP Antibody, GFP, Green Fluorescent Pro
• Note: For research use only
• Application notes: Anti-GFP is designed to detect GFP and its variants. Goat Anti-GFP has been tested by ELISA, SDS-PAGE, Western blot, and Immunofluorescence. This product is ideal for western blotting, ELISA, immunofluorescence, IHC, and IP. This antibody can be used to detect GFP by ELISA (sandwich or capture) for the direct binding of antigen and recognizes wild type, recombinant and enhanced forms of GFP. Biotin conjugated polyclonal anti-GFP used in a sandwich ELISA is well suited to titrate GFP in solution using this antibody in combination with Rockland's monoclonal anti-GFP (600-301-215) using either form of the antibody as the capture or detection antibody. However, use the monoclonal form only for the detection of wild type or recombinant GFP as this form does not sufficiently detect 'enhanced' GFP. The detection antibody is typically conjugated to biotin and subsequently reacted with streptavidin-HRP (code # S000-03). Fluorochrome conjugated polyclonal anti-GFP can be used to detect GFP by immunofluorescence microscopy in prokaryotic (E.coli) and eukaryotic (CHO cells) expression systems and detects GFP containing inserts. Significant amplification of signal is achieved using fluorochrome conjugated polyclonal anti-GFP relative to the fluorescence of GFP alone. For immunoblotting use either alkaline phosphatase or peroxidase conjugated polyclonal anti-GFP to detect GFP or GFP-containing proteins on western blots. Researchers should determine optimal titers for applications.
• Expiration Date: 12 months from date of receipt.

References

Meine, Rosanna et al. Indole-3-Carbonitriles as DYRK1A Inhibitors by Fragment-Based Drug Design Molecules, 23, E64 (2018)

PubMed: 29364148

Binding sites for transcription factors with predicted pioneer function co-occur with LHX2 peaks. (a, b) Hierarchical clustering of LHX2 ChIP-Seq regulatory motifs and assigned representative logos are represented at E14 (a) and P2 (b) (linkage = average; similarity threshold cor = 0.6, ncor = 0.4, w = 5). The most enriched cluster comprises LHX2 and multiple variations of the same motif. (c, d) Known transcription factor motifs preferentially enriched in either E14 or P2 LHX2 ChIP-Seq peaks and identified by pairwise comparison are shown. Percentage of motif occurrences are reported for input and background datasets. The relative expression level of the corresponding transcription factor mRNA at E14/P2 is indicated by the blue/red color gradient, respectively.

Binding sites for transcription factors with predicted pioneer function co-occur with LHX2 peaks. a, b Hierarchical clustering of LHX2 ChIP-Seq regulatory motifs and assigned representative logos are represented at E14 (a) and P2 (b) (linkage = average; similarity threshold cor = 0.6, ncor = 0.4, w = 5). The most enriched cluster comprises LHX2 and multiple variations of the same motif. (c, d) Known transcription factor motifs preferentially enriched in either E14 or P2 LHX2 ChIP-Seq peaks and identified by pairwise comparison are shown. Percentage of motif occurrences are reported for input and background datasets. The relative expression level of the corresponding transcription factor mRNA at E14/P2 is indicated by the blue/red color gradient.

ELISA results of purified Goat Anti-GFP Antibody mx3. Each well was coated in 1.0 µg of antigen GFP [Red Line], human IgG [Green Line], Mouse IgG [Blue Line], and Rat IgG [Purple Line]. The starting dilution of antibody was 5 µg/mL and the X-axis represents the Log10 of a 3-fold dilution. The titer is 1:14800. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using Buffer, Substrate, and Donkey Anti-Goat IgG Antibody HRP (p/n orb347032).

Immunofluorescence Microscopy of Anti-GFP (GOAT) Antibody. Tissue: E5.5 Hex-GFP transgenic mouse embryo. Primary antibody: Goat anti-GFP was used at 1:500 dilution. Secondary antibody: Fluorchrome conjugated Anti-goat IgG secondary antibody at 1:10000 for 45 min at RT. Staining: GFP as green fluorescent signal with DAPI blue counterstain.

Immunofluorescence Microscopy of GFP-GOAT-Antibody. Tissue: Sf-1:Cre mice crossed to the Z/EG reporter line. Mouse brain (coronal view, 20X magnification). Fixation: 4% PFA/PBS with o/n fixation, and subsequently transferred to a 30% sucrose solution. Antigen retrieval: frozen in OCT freezing medium (Sakura) and cryostat sectioned at 40 microns. Primary antibody: Goat anti-GFP was used at 1:500 dilution in free floating imunnohistochemistry to detect GFP. Secondary antibody: Fluorchrome conjugated Anti-goat IgG secondary antibody was used for detection at 1:500 at 1:10000 for 45 min at RT. Localization: Sf-1+ neurons and their processes of the ventromedial nucleus of the hypothalamus. Staining: eGFP as green fluorescent signal and sections were counterstained with DAPI.

Immunofluorescence Microscopy of GFP-GOAT-Antibody. Tissue: Sf-1:Cre mice crossed to the Z/EG reporter line. Mouse brain (coronal view, 20X magnification). Fixation: 4% PFA/PBS with o/n fixation, and subsequently transferred to a 30% sucrose solution. Antigen retrieval: frozen in OCT freezing medium (Sakura) and cryostat sectioned at 40 microns. Primary antibody: Goat anti-GFP was used at 1:500 dilution in free floating imunnohistochemistry to detect GFP. Secondary antibody: Fluorchrome conjugated Anti-goat IgG secondary antibody was used for detection at 1:500 at 1:10000 for 45 min at RT. Localization: Sf-1+ neurons and their processes of the ventromedial nucleus of the hypothalamus. Staining: eGFP as green fluorescent signal and sections were counterstained with DAPI.

Increased nestin+ cells in prostate stroma are a subset of mesenchymal/stromal stem cells. (A): Immunofluorescence analysis of prostate tissue sections from Nestin‐GFP transgenic mice at 0, 1, 2, and 4 weeks after initial PE injection using antibody against GFP. Arrows indicate GFP+ cells. Bar graph on the right shows the quantitative analyses of the ratio of GFP+ cells to total cells. Scale bar = 50μm. (B): Double‐immunohistofluorescence analysis of prostate tissue sections from Nestin‐GFP mice using antibodies against GFP and CD90 or Sca1. Scale bar = 20 μm. (C): Flow cytometry analysis of GFP+ CD45–cells (percentage of total cells counted) in blood of Nestin‐GFP mice at indicated time points after PE injection. (D): Flow cytometry analysis of CD90+ Sca1+ CD11b–CD45– cells (percentage of total cells counted) in blood of wild‐type mice at indicated time points after PE injection. (E): Fluorescence‐activated cell sorting of GFP+ CD45−cells from ventral prostate tissue of 6‐week‐old Nestin‐GFP mice. (F): Single CFU‐F‐derived GFP+ CD45− cell strains were isolated and individually expanded for characterization assays. (G): Single GFP+ CD45− clones were isolated, expanded, and cultured. Cells were separately incubated with fibrogenic media, osteogenic media, adipogenic media, and chondrogenic media, and they were stained for vimentin (upper left panel), Alizarin Red S (upper right panel), Oil Red O (lower left panel), and Toluidine Blue (lower right panel), respectively. (H): Double‐immunohistofluorescence analysis of prostate tissue sections from 6‐week‐old Nestin‐Cre, Rosa26‐YFPflox/+ mice using antibodies against YFP (green) and vimentin (red, top panels) or α‐SMA (red, bottom panels). Scale bar = 50μm. n=4–5 per group. Data are shown as the mean ± SD. ∗, p <.05, ∗∗, p <.01 determined by multifactorial analysis of variance. Abbreviations: CFU‐F, colony‐forming unit fibroblast; DAPI, 4', 6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; PE, phenylephrine; α‐SMA, α‐smooth muscle actin; w, weeks.

Increased nestin+ cells in prostate stroma are a subset of mesenchymal/stromal stem cells. (A): Immunofluorescence analysis of prostate tissue sections from Nestin‐GFP transgenic mice at 0, 1, 2, and 4 weeks after initial PE injection using antibody against GFP. Arrows indicate GFP+ cells. Bar graph on the right shows the quantitative analyses of the ratio of GFP+ cells to total cells. Scale bar = 50μm. (B): Double‐immunohistofluorescence analysis of prostate tissue sections from Nestin‐GFP mice using antibodies against GFP and CD90 or Sca1. Scale bar = 20 μm. (C): Flow cytometry analysis of GFP+ CD45–cells (percentage of total cells counted) in blood of Nestin‐GFP mice at indicated time points after PE injection. (D): Flow cytometry analysis of CD90+ Sca1+ CD11b–CD45– cells (percentage of total cells counted) in blood of wild‐type mice at indicated time points after PE injection. (E): Fluorescence‐activated cell sorting of GFP+ CD45−cells from ventral prostate tissue of 6‐week‐old Nestin‐GFP mice. (F): Single CFU‐F‐derived GFP+ CD45− cell strains were isolated and individually expanded for characterization assays. (G): Single GFP+ CD45− clones were isolated, expanded, and cultured. Cells were separately incubated with fibrogenic media, osteogenic media, adipogenic media, and chondrogenic media, and they were stained for vimentin (upper left panel), Alizarin Red S (upper right panel), Oil Red O (lower left panel), and Toluidine Blue (lower right panel), respectively. (H): Double‐immunohistofluorescence analysis of prostate tissue sections from 6‐week‐old Nestin‐Cre, Rosa26‐YFPflox/+ mice using antibodies against YFP (green) and vimentin (red, top panels) or α‐SMA (red, bottom panels). Scale bar = 50μm. n=4–5 per group. Data are shown as the mean ± SD. ∗, p <.05, ∗∗, p <.01 determined by multifactorial analysis of variance. Abbreviations: CFU‐F, colony‐forming unit fibroblast; DAPI, 4', 6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; PE, phenylephrine; α‐SMA, α‐smooth muscle actin; w, weeks.

LHX2 coordinates variations in chromatin accessibility at the Sox2 locus. Custom tracks of E14 and P2 LHX2 ChIP-Seq reads and age-matched ATAC-Seq from purified RPCs and post mitotic precursors were configured on the mm10 UCSC murine genome assembly. Regions of interest targeted by LHX2, where variations by RNA-Seq and/or ATAC-Seq coverage are observed in Lhx2 cKO retinas are highlighted. Association with H3K27ac-labeled enhancer elements is also indicated. The LHX2-coordinated regulatory module is defined as follows: regulatory regions that display variations in ATAC-Seq coverage without a corresponding variation in the nearest transcript by E14 Lhx2 cKO RNA-Seq are putatively assigned (arrows) to the nearest gene exhibiting variation in transcript levels.

LHX2 regulates cell cycle genes and the Notch signaling pathway by targeting promoters and non-coding elements in nucleosome free regions associated with active enhancers. a LHX2 motif density around the centers of ChIP-Seq peaks at E14 and P2. b Enrichment of LHX2 ChIP-Seq peaks distributed across different genomic regions (log2 fold enrichment). c–f Percentage of LHX2 target genes in retinal term assigned to at least one age-matched LHX2 ChIP-Seq peak. Enrichment was computed between target genes in term and total number of known genes in term for peaks shared at E14 and P2 (c), stage-specific peaks (d), and all peaks detected at E14 (e) and P2 (f). RNA-Seq from age-matched Lhx2 cKO retinas identifies Lhx2-dependent genes sets. (*p-value < 0.05, ***p-value < 0.001). Asterisks in parenthesis refer to p-values before Bonferroni–Hochberg correction. g, h Heatmaps of raw reads from LHX2 ChIP-Seq peaks are plotted across nucleosome centered regions from age-matched ATAC-Seq samples. Each row represents a 3 kb window centered at maximum read pile-up. LHX2 motifs co-localize with RNA-Seq raw reads. Meta-profiles of the class II enhancer-associated H3K27ac marks were compiled at bidirectionally transcribed regions from the E14 (g) and P2 RPCs fractions (h) (background in gray, opposite strands replicates in hue). i, j Functional enrichment of Lhx2-dependent genes sets encoded within open chromatin regions by binomial distribution.

LHX2 regulates cell cycle genes and the Notch signaling pathway by targeting promoters and non-coding elements in nucleosome free regions associated with active enhancers. A) LHX2 motif density around the centers of ChIP-Seq peaks at E14 and P2. B) Enrichment of LHX2 ChIP-Seq peaks distributed across different genomic regions (log2 fold enrichment). (C-F) Percentage of LHX2 target genes in retinal term assigned to at least one age-matched LHX2 ChIP-Seq peak. Enrichment was computed between target genes in term and total number of known genes in term for peaks shared at E14 and P2 (C), stage-specific peaks (D), and all peaks detected at E14 (E) and P2 (F). RNA-Seq from age-matched Lhx2 cKO retinas identifies Lhx2-dependent genes sets. (*p-value < 0.05, ***p-value < 0.001). Asterisks in parenthesis refer to p-values before Bonferroni–Hochberg correction. (G, H) Heatmaps of raw reads from LHX2 ChIP-Seq peaks are plotted across nucleosome centered regions from age-matched ATAC-Seq samples. Each row represents a 3 kb window centered at maximum read pile-up. LHX2 motifs co-localize with RNA-Seq raw reads. Meta-profiles of the class II enhancer-associated H3K27ac marks were compiled at bidirectionally transcribed regions from the E14 (G) and P2 RPCs fractions (H) (background in gray, opposite strands replicates in hue). (I, J) Functional enrichment of Lhx2-dependent genes sets encoded within open chromatin regions by binomial distribution.

Maximal activity of HIPKs depends on the activation loop tyrosine. Wild type GFP-HIPK fusion proteins and the respective Tyr→Phe mutants were immunoprecipitated from HeLa cells and subjected to kinase assays with recombinant GST-p27Kip1(A), myelin basic protein (B) or DYRKtide (C). GFP served as background control. A, Phosphorylation of p27Kip1 at Ser10 was detected by immunoblotting with a phosphorylation-specific antibody. For quantitative evaluation, pSer10 immunoreactivity was normalised to GFP immunoreactivity, which reflects the amount of kinase in the reaction. The blots illustrate a representative experiment, and the relative catalytic activities as determined from 3–4 assays are shown below the panels (means ± SD). One-sample t test: *, p < 0.05; **, p < 0.01. B and C, Phosphorylation of MBP and DYRKtide was measured in triplicate as incorporation of 32P. Background values from the GFP control samples were subtracted and activities were normalised to the amount of kinase in the reaction as determined by GFP immunoreactivity. Column diagrams illustrate catalytic activities relative to HIPK2 (WT). The results were replicated in independent experiments, except for a missing value of HIPK1.

MSCs are traced in special organized regions. MSC regions and normal regions in regenerated liver tissue of cirrhotic mice treated with pHx + 2 x 106 MSC stained for Haematoxylin‐eosin, GFP and α‐SMA (40x magnifications, MSCs are indicated by the black arrows). GFP, green fluorescent protein; α‐SMA, smooth muscle actin; MSCs, mesenchymal stromal cells; pHx, partial hepatectomy figure.

Multi-lysate Western Blot of Goat anti-GFP antibody. Marker: Opal Pre-stained ladder. Lane 1: HEK293 lysate (p/n orb348669). Lane 2: HeLa Lysate (p/n orb348668). Lane 3: CHO/K1 Lysate. Lane 4: MDA-MB-231 (p/n orb348700). Lane 5: A431 Lysate (p/n orb348665). Lane 6: Jurkat Lysate (p/n orb348674). Lane 7: NIH/3T3 Lysate (p/n orb348714). Lane 8: E-coli HCP Control (p/n orb342262). Lane 9: FLAG Positive Control Lysate (p/n orb348661). Lane 10: Red Fluorescent Protein (p/n orb345960). Lane 11: Green Fluorescent Protein (p/n orb345957). Lane 12: Glutathione-S-Transferase Protein (p/n orb345956). Lane 13: Maltose Binding Protein. Load: 10 µg of lysate or 50 ng of purified protein per lane. Primary antibody: GFP antibody at 1 ug/ml overnight at 4C. Secondary antibody: Peroxidase goat secondary antibody at 1:30000 for 60 min at RT. Blocking Buffer: 1% Casein-TTBS for 30 min at RT. Predicted/Observed size: 30 kDa for GFP.

Npas4 expression during neural differentiation of ESCs. (A) RT-PCR analysis was used to determine the temporal expression profile of Npas4 mRNA in relation to various marker genes during N2B27 differentiation of mESCs (n=3). Primers to the reference gene β-actin were used as a loading control. The negative control reaction (−) contained water in place of template cDNA. (B) The changes in Npas4 expression during N2B27 differentiation were quantified using qRT-PCR. At each time point Npas4 expression was normalized to β-actin expression and fold changes are relative to Day 0 (undifferentiated mESCs). Mean values and standard deviations of three independent experiments (n=3) are displayed. (C)In situ hybridization analysis of Npas4 mRNA expression at Day 4 of N2B27 differentiation of mESCs. Representative images of differentiating colonies from two independent experiments (n=2) are shown. Top panel - Npas4 antisense probe; Bottom panel - Npas4 sense probe. Scale bar = 100 μm. (D) Immunoblotting was used to determine the temporal expression profile of the Npas4 protein during N2B27 differentiation of mESCs. An antibody to the reference protein β-actin was used as a loading control (n=3). (E) Immunoblotting was used to determine the temporal expression profile of Sox1 in the 46C cell line (using an antibody to GFP) and βIII tubulin during N2B27 differentiation of mESCs. An antibody to the reference protein β-actin was used as a loading control (n=3). (F) RT-PCR analysis of NPAS4 mRNA in relation to various marker genes during Noggin-induced neural differentiation of hESCs (n=3). Primers to the reference gene β-ACTIN were used as a loading control. The negative control reaction (−) contained water in place of template cDNA. hESCs, human embryonic stem cells; mESCs, mouse embryonic stem cells; NS, neurospheres.

P2X7-EGFP expression in retina, sciatic nerves, spinal cord, and at the neuromuscular synapse. (A) EGFP exclusively co-localizes with microglia and endothelial cells in the adult mouse retina. Upper panel: Middle, retinal slice labeled for GFP (orb345286, Biorbyt), Iba1 (marker for microglia/macrophages) and glutamine synthetase (marker for Müller glia). Left and right, retinal flat mounts scanned at the plane of the ganlion cell layer (GCL) and outer plexiform layer (OPL), respectively, to delineate microglia residing in these retinal layers. Astrocytes in the GCL were labeled with GFAP. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. Lower panel: Co-staining of EGFP with neuronal marker PKCα (left) and glutamine synthetase (two right panels) at higher contrast and resolution to show absence of neuronal P2X7-EGFP. Cell nuclei were counterstained with Hoechst 33342 (blue) Scale bars: 20 µM. n=2 individual line 61 in FVB/C57b/6 hybrid mice.

Pairwise comparison of ATAC-Seq data identifies open chromatin regions from early and late RPCs, with a broad overrepresentation of LHX2 related motifs. A) Workflow for epigenomic profiling of RPC. B) Venn-diagram represents open chromatin regions identified in early- and late-stage VSX2 (CHX10)—GFP-positive RPCs. C) Hierarchical clustering of known motifs in the vertebrate genome. Inset represents the major cluster of probabilistically assigned position weight matrices (PWMs) identified in open chromatin regions from early- and late-stage RPCs (scale = 1 node/pixel) (linkage = average; similarity threshold cor = 0.6, ncor = 0.4, w = 5) (Lhx2 instances as blue pixel strokes). D) Relative RNA-Seq expression for homeobox transcription factors. E) Representative LHX2 logos (MA0700.1, k-mer sig = 300, e-value = 1e-300), with positional variations of the same motif instance. F) Known motifs enrichment in open chromatin regions from early and late RPCs by binomial scoring of PWMs. G) LHX2 footprints from open chromatin regions identified in early- and late-stage RPCs. H) Custom tracks of RPC-derived ATAC-Seq profiles and aged-matched LHX2 ChIP-Seq feature the Vsx2 locus (mm9).

Pairwise comparison of ATAC-Seq data identifies open chromatin regions from early and late RPCs, with a broad overrepresentation of LHX2 related motifs. A) Workflow for epigenomic profiling of RPC. B) Venn-diagram represents open chromatin regions identified in early- and late-stage VSX2 (CHX10)—GFP-positive RPCs. C) Hierarchical clustering of known motifs in the vertebrate genome. Inset represents the major cluster of probabilistically assigned position weight matrices (PWMs) identified in open chromatin regions from early- and late-stage RPCs (scale = 1 node/pixel) (linkage = average; similarity threshold cor = 0.6, ncor = 0.4, w = 5) (Lhx2 instances as blue pixel strokes). D) Relative RNA-Seq expression for homeobox transcription factors. E) Representative LHX2 logos (MA0700.1, k-mer sig = 300, e-value = 1e-300), with positional variations of the same motif instance. F) Known motifs enrichment in open chromatin regions from early and late RPCs by binomial scoring of PWMs. G) LHX2 footprints from open chromatin regions identified in early- and late-stage RPCs. H) Custom tracks of RPC-derived ATAC-Seq profiles and aged-matched LHX2 ChIP-Seq feature the Vsx2 locus (mm9).

Pairwise comparison of ATAC-Seq data identifies open chromatin regions from early and late RPCs, with a broad overrepresentation of LHX2 related motifs. A) Workflow for epigenomic profiling of RPC. B) Venn-diagram represents open chromatin regions identified in early- and late-stage VSX2 (CHX10)—GFP-positive RPCs. C) Hierarchical clustering of known motifs in the vertebrate genome. Inset represents the major cluster of probabilistically assigned position weight matrices (PWMs) identified in open chromatin regions from early- and late-stage RPCs (scale = 1 node/pixel) (linkage = average; similarity threshold cor = 0.6, ncor = 0.4, w = 5) (Lhx2 instances as blue pixel strokes). D) Relative RNA-Seq expression for homeobox transcription factors. E) Representative LHX2 logos (MA0700.1, k-mer sig = 300, e-value = 1e-300), with positional variations of the same motif instance. F) Known motifs enrichment in open chromatin regions from early and late RPCs by binomial scoring of PWMs. G) LHX2 footprints from open chromatin regions identified in early- and late-stage RPCs. H) Custom tracks of RPC-derived ATAC-Seq profiles and aged-matched LHX2 ChIP-Seq feature the Vsx2 locus (mm9).

Subcellular distribution of HIPK mutants. COS-7 cells were transfected with expression vectors for GFP-HIPK1 (A), GFP-HIPK2 (B), GFP-HIPK3 (C) or GFP-HIPK4 (D). The cellular localisation of the HIPK constructs was evaluated by imaging GFP autofluorescence in relation to bisbenzimide-stained nuclei. For each kinase, cells were classified into 3 major patterns that are illustrated by representative images and icons. The cells shown were transfected with (top to bottom) HIPK1 WT, DN, YF; HIPK2 WT, WT, YF; HIPK3 WT, WT, DN and HIPK4 DN, DN.

Tyrosine phosphorylation of HIPK mutants. HeLa cells were transiently transfected with the indicated expression constructs for HIPK1 (A), HIPK2 (B), HIPK3 (C) or HIPK4 (D). Sodium orthovanadate (Na3VO4) was added to every second sample for 1 h before lysis. GFP fusion proteins were immunoprecipitated and analysed by immunodetection with antibodies for pTyr and GFP. The panels are representative of 2–3 independent experiments.

Tyrosine phosphorylation of HIPK mutants. HeLa cells were transiently transfected with the indicated expression constructs for HIPK1 (A), HIPK2 (B), HIPK3 (C) or HIPK4 (D). Sodium orthovanadate (Na3VO4) was added to every second sample for 1 h before lysis. GFP fusion proteins were immunoprecipitated and analysed by immunodetection with antibodies for pTyr and GFP. The panels are representative of 2–3 independent experiments.

Tyrosine phosphorylation of HIPK mutants. HeLa cells were transiently transfected with the indicated expression constructs for HIPK1 (A), HIPK2 (B), HIPK3 (C) or HIPK4 (D). Sodium orthovanadate (Na3VO4) was added to every second sample for 1 h before lysis. GFP fusion proteins were immunoprecipitated and analysed by immunodetection with antibodies for pTyr and GFP. The panels are representative of 2–3 independent experiments.

Tyrosine phosphorylation of HIPK mutants. HeLa cells were transiently transfected with the indicated expression constructs for HIPK1 (A), HIPK2 (B), HIPK3 (C) or HIPK4 (D). Sodium orthovanadate (Na3VO4) was added to every second sample for 1 h before lysis. GFP fusion proteins were immunoprecipitated and analysed by immunodetection with antibodies for pTyr and GFP. The panels are representative of 2–3 independent experiments.

Upregulation of P2X7 expression in postischemic retinae 3 days post injury (dpi) of wt and P2X7-EGFP transgenic animals. (A) Retinal flat mount scanned at the plane of the ganglion cell layer (GCL) to delineate microglia residing in this retinal layer. Co-labeling for GFP (orb345286, Biorbyt), Iba1 (marker for microglia/macrophages) and glutamine synthetase (marker for Müller glia) shows that EGFP exclusively co-localizes with microglia and in the postischemic retina also with morphologically distinct Iba1-positive cells that likely correspond to infiltrated macrophages. Note unspecific vessel staining caused by the secondary antibody against glutamine synthetase. Scale bars: 20 µM. n=2 individual line 61 in FVB/C57b/6 hybrid mice. (B) Direct EGFP-fluorescence captured at identical microscope settings in non-fixed retinal flatmounts focused on the GCL shows increased EGFP signal in vessels and microglia and an increase in the number of microglia and/or macrophages. Scale bar: 20 µM. (C) P2X7 expression determined by quantitative real-time PCR on the indicated cell types isolated by immunomagnetic separation from control and postischemic (3 dpi) retinae of line 17 P2X7-EGFP transgenic mice in (C57b/6 background). Bars represent mean ±SEM and include data from 3 to 4 animals/each genotype/condition. Significance between expression level in the untreated control eye of the respective genotype was analyzed using unpaired two-tailed Mann-Whitney-U-test and indicated as: *P< 0.05. Data were normalized to the housekeeper pyruvate dehydrogenase beta (PDHB) and results are presented as relative expression levels compared to that in microglia of healthy wild-type retinae. Increase in P2X7 signal in RPE is most likely due to contamination by infiltrating immune cells.

Western Blot of Goat anti-GFP antibody with lysates. Lane 1: Opal Prestained Molecular Weight Marker. Lane 2: GFP/HEK293T WCL [0.05/10 µg]. Lane 3: HEK293T WCL [10 µg]. Lane 4: GFP/NIH-3T3 WCL (p/n orb345957/orb348714) [0.05/10 µg]. Lane 5: NIH-3T3 WCL (p/n orb348714) [10 µg]. Lane 6: GFP/PC-12 WCL (p/n orb345957/orb348733) [0.05/10 µg]. Lane 7: PC-12 WCL (p/n orb348733) [10 µg]. Primary antibody: Anti-GFP antibody at 1 ug/ml overnight at 2-8°C. Secondary antibody: Donkey Anti-Goat IgG HRP secondary antibody (p/n orb347031) at 1:40000 for 30 min at RT. Blocking Buffer: BlockOut Buffer (p/n orb348644) for 1 hr at RT. Predicted/Observed size: ~27 kDa for GFP.

Western Blot of Goat anti-GFP Antibody with Serums. Lane 1: Opal Prestained Molecular Weight Marker. Lane 2: GFP/Human Serum [0.01/0.02 µL] [+]. Lane 3: Human Serum [0.02 µL] [-]. Lane 4: GFP/Mouse Serum [0.01/0.02 µL] [+]. Lane 5: Mouse Serum [0.02 µL] [-]. Lane 6: GFP/Rat Serum [0.01/0.02 µL] [+]. Lane 7: Rat Serum [0.02 µL] [-]. Primary antibody: Anti-GFP antibody at 1.0 ug/ml overnight at 2-8°C. Secondary antibody: Donkey Anti-Goat IgG HRP secondary antibody at 1:40000 for 30 min at RT. Blocking Buffer: BlockOut Buffer (p/n orb348644) for 1 hr at RT. Predicted/Observed size: ~27 kDa for GFP.

Western Blot of Goat anti-GFP antibody. Lane 1: HeLa cells. Lane 2: mock transfected HeLa cell lysate. Load: 35 µg per lane. Primary antibody: GFP antibody at 1 µg/ml for 1 h at room temperature. Secondary antibody: IRDye® 800 conjugated Donkey-a-Goat IgG [H&L] MX7 secondary antibody at 1:2500 for 45 min at RT. Block: 5% BLOTTO overnight at 4°C. Predicted/Observed size: 27 kDa, 33 kDa for GFP. Other band(s): none.