Anti GFP

ELISA results of purified Goat Anti GFP Antibody mx3. Each well was coated in 1.0 ug 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 ug/mL and the X axis represents the Log10 of a 3 fold dilution. The titer is 1:14,800. This titration is a 4 parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using Buffer (p/n MB 060 1000), Substrate (p/n TMB 8000), and Donkey Anti Goat IgG Antibody HRP (p/n 605 703 125).

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 (600-101-215, Rockland), Iba1 (marker for microglia/macrophages) and glutamine synthetase (marker for Muller 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 um. 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 um. (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. Figure provided by CiteAb. Source: Elife, PMID: 30074479.

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 (600-101-215, Rockland), Iba1 (marker for microglia/macrophages) and glutamine synthetase (marker for Muller 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 um. n = 2 individual line 61 in FVB/C57b/6 hybrid mice. Figure provided by CiteAb. Source: Elife, PMID: 30074479.

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 Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.

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 (Supplementary Table？4 and Supplementary Data？1) 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 (target genes populating the enriched ontologies are in Supplementary Data？2). (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 motif occurrence in open chromatin regions is displayed and reported in Supplementary Tables？6. 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 (Supplementary Tables？6) by binomial distribution Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.

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 Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.

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 Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.

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 (Supplementary Table？4 and Supplementary Data？1) 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 (target genes populating the enriched ontologies are in Supplementary Data？2). 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 motif occurrence in open chromatin regions is displayed and reported in Supplementary Tables？6. 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 (Supplementary Tables？6) by binomial distribution Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.

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 (left, Jaspar 2016 non-redundant vertebrates core). 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). Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.

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 (left, Jaspar 2016 non-redundant vertebrates core). 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). Figure provided by CiteAb. Source: Commun Biol, PMID: 31044167.