Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Expression qPCR analysis for HIF-2α mRNA in tissue lysates from renal tumors or corresponding normal kidney tissue from the Erlangen RCC cohort (ccRCC: clear cell renal carcinoma [ n = 114], pRCC: papillary renal cell carcinoma [ n = 16], chRCC: chromophobe renal cell carcinoma [ n = 11]). Data shown are median and interquartile range. Significance was tested by two-way ANOVA followed by Bonferroni’s post hoc test. b RNA-seq expression values for HIF-2α in different renal tumors and corresponding normal kidney tissue from the TCGA KIRC ( n = 72), KIRP ( n = 31) and KICH ( n = 23) data sets. Median and interquartile range. P -values were determined by two-way ANOVA followed by Bonferroni’s post hoc test. c RNAscope experiment for HIF-2α mRNA in ccRCC tissue. Arrows indicate HIF-2α mRNA transcripts in tumor cells. V = vessel. Scale bars, 100 µm or 75 µm as indicated. Representative image from independent samples of seven different patients with similar results. d Log 2 fold change of HIF-related genes in snRNA-seq data comparing tumor cells from 30 ccRCC samples versus proximal tubule (PT) cells from four adjacent kidney tissue samples . Only samples with significantly differentially expressed transcripts are shown (adjusted p < 0.05, average log 2 fold change ≠ 0). e Isolation of renal tumor cells and corresponding tubule cells from non-diseased tissue of tumor nephrectomy specimens. Created in BioRender. Naas, S. (2025) https://BioRender.com/c87s173 . f Immunoblot analyses for HIF-1α, HIF-2α, pVHL, and β-actin in lysates from primary tubule (PTC) and tumor (ccRCC) cells isolated from tissue specimens of patient #1. Representative blot from 19 independent experiments with similar results. g Expression qPCR analysis for HIF-2α mRNA in lysates from primary tubule cells (PTC) and primary tumor (ccRCC) isolated from 34 different individuals. Graph shows median with interquartile range and p -values from a two-tailed unpaired t-test.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Expressing, RNA Sequencing, RNAscope, Isolation, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Sequencing tracks from ATAC- and H3K27ac ChIP-seq experiments performed in cells from patient #1 at the EPAS1 locus. Activity-by-contact (ABC) analysis was used to predict enhancer-promoter interactions. Additionally, tracks from published data from Capture-C experiments in 786-0 cells , gained enhancer activity in ccRCC (ccRCC-enhancer) , ccRCC cancer-cell specific differentially accessible regions (ccRCC-specific DACR) as determined by snATAC-seq , KIRC hypomethylated CpGs (Beta-value cutoff 0.25, adjusted p -value cutoff 0.01) and KIRC-specific ATAC-seq elements are shown . b Differentially active enhancer regions on chromosome 2 in ccRCC tumors from Yao et al . Average log 2 fold chance (H3K27ac signal, ten tumors vs corresponding normal tissue) and -log 10 adjusted p -value are shown. Significantly regulated elements (adjusted p < 0.05, light blue) that overlap KIRC-specific ATAC sites are depicted in dark blue. Sites highlighted in red overlap KIRC-specific sites, show a positive log 2 fold change and are localized within 5 Mb of the EPAS1 gene. Enhancer enh-1031503 covers elements KIRC_10767-KIRC_10770. For comparison, the H3K27ac signal at the EPAS1 promoter is marked, but not significantly regulated. c ATAC-seq tracks for the different TCGA tumor entities at the ccRCC-enhancer site with gained activity in ccRCC . KIRC specific ATAC-seq sites 10767-70 are indicated above the tracks. d Spearman’s rank correlation analysis of HIF-2α mRNA expression (TPM: transcripts per million) and accessibility at KIRC_10770 in the KIRC data set. Values are from samples for which both analyses were available ( n = 16 different tumors). Gray region depicts 95% confidence interval. Significance was assessed with a two-sided t-approximation test.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Sequencing, ChIP-sequencing, Activity Assay, Capture-C, Comparison, Expressing

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a RSEM-normalized RNA-seq values for HIF-2α in the TCGA KIRC cohort stratified for VHL copy number alterations (diploid n = 38, deletion n = 309). P -values were determined by an unpaired, two-tailed t-test. b RT-qPCR analyses for HIF-2α mRNA in RCC4 cells with or without functional pVHL. Graph shows mean + SD. P -values were determined by a two-tailed one sample t-test with a hypothetical value of 1. n = 4 independent experiments. c Representative immunoblot ( n = 2) for HIF-1α, HIF-2α, hemagglutinin (HA), and β-actin in lysates from RCC4/i.VHL-HA cells exposed to 0.5 µg/ml doxycycline to induce pVHL for the indicated time. d RT-qPCR analyses for HIF-2α mRNA in lysates from RCC4/i.VHL-HA cells treated with 0.5 µg/ml doxycycline for the indicated time. Expression values are mean + SD from three independent experiments. Significance was tested by a two-tailed one sample t-test with a hypothetical value of 1. e Expression qPCR analysis for HIF-2α mRNA in lysates from RCC4 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. Mean + SD from 7 independent experiments. Differences were assessed by a two-tailed one sample t-test with a hypothetical value of 1. f RT-qPCR analysis for HIF-2α mRNA in lysates from 786-0 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. Graph shows mean + SD from 5 independent experiments. P -values were determined by a two-tailed one sample t-test with a hypothetical value of 1. g Representative immunoblot ( n = 2) for HIF-2α, HIF-1α, HIF-1β, and β-actin from lysates of RCC4 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. h Representative immunoblot ( n = 2) for HIF-2α, HIF-1β, and β-actin from lysates of 786-0 cells transfected with sgRNA targeting HIF-1β or non-targeting (nt) control using CRISPR/Cas9. i ATAC-seq tracks at the ccRCC-activated enhancer in RCC4/i.VHL-HA cells as well as single clones of HIF-1β knock-out or nt control RCC4 or 786-0 cells, respectively. RCC4/i.VHL-HA were exposed either to 0.5 µg/ml doxycycline (Dox +) or to DMSO control (Dox -) for 18 h. The ATAC element KIRC_10770 is highlighted in orange. j H3K27ac CUT&Tag tracks from the same cells at the same genomic region as in i ).

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: RNA Sequencing, Two Tailed Test, Quantitative RT-PCR, Functional Assay, Western Blot, Expressing, Transfection, Control, CRISPR, Clone Assay, Knock-Out

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Expression qPCR analyses for HIF-2α mRNA in lysates from 105 isolates of primary tubule cells (PTC) exposed to 1 mM DMOG or control conditions for 16 h. Values are mean +/− SD from n = 105 independent experiments. Unpaired, two-tailed t-test. b Expression qPCR for HIF-2α mRNA in lysates from PTC depleted for HIF-1α or HIF-1β using siRNA and stimulated with or without 1 mM DMOG for 16 h. Non-targeting (nt) control siRNA was used as control. Values are mean + SD. Significance was tested by two-way ANOVA followed by Bonferroni’s post hoc test. Data is from nine independent experiments using PTC from nine different individuals. c HIF ChIP-seq tracks from lysates of primary tubule cells (PTC, green) treated with 1 mM DMOG for 4 h and ccRCC cells (blue) at the PRKCE-EPAS1 locus. ChIP-seq track for the activity marker H3K27ac in isolated ccRCC cells from patient #1 and hypomethylated CpGs in TCGA KIRC are also shown. KIRC-specific polymorphisms identified by the Cis-eQTL PancanQTL analysis as an eQTL for EPAS1 expression are indicated . Germline variants associated with renal cancer development within the EPAS1 -coding region are shown (RCC risk SNP) , . d Correlation of rs12617313 genotype and HIF-2α mRNA expression in the KIRC TCGA normal tissue and tumor data. Number of samples for normal tissue: Genotype TT n = 19, AT n = 25, AA n = 16. Number of samples for tumor tissue (KIRC): Genotype TT n = 94, AT n = 188, AA = 121. Data shown are median and interquartile range. χ 2 -test; p -value as indicated for higher expression in AA individuals compared with TT individuals. e Genotype-expression correlation for rs12617313 and HIF-2α mRNA in PTC exposed to 1 mM DMOG for 16 h or left untreated. Number of samples for each genotype: TT n = 26, AT n = 58, AA n = 21. Data shown are median and interquartile range. Significance was tested by two-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Expressing, Control, Two Tailed Test, ChIP-sequencing, Activity Assay, Marker, Isolation

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a ChIP-seq tracks for HIF-1β, PAX8 , H3K27ac and HNF-1β CUT&Tag-seq track from 786-0 cells at EPAS1 -enhancer E2. Binding sites for HNF-1β (E2_HNF-1β) and PAX8 (E2_PAX8) within KIRC_10767 are highlighted in blue. HIF-binding site is marked in orange. b Expression qPCR analyses for HIF-2α mRNA in lysates from 786-0 cells depleted for the indicated transcription factor (HNF-1β or PAX8). Values are mean + SD from 5 (HNF-1β) or 4 (PAX8) independent experiments. Statistical significance was assessed by a two-tailed one sample t-test with a hypothetical value of 1. c Immunoblot analyses for HIF-2α, HNF-1β, and β-actin in lysates from 786-0 cells depleted for HNF-1β using CRISPR/Cas9. Representative blot from two independent experiments with similar results. d Immunoblot analyses for HIF-2α, PAX8, and β-actin in lysates from 786-0 cells depleted for PAX8 using CRISPR/Cas9. Representative blot from two independent experiments with similar results. e ATAC-seq and CUT&Tag-seq tracks of the chromatin activity marker H3K27ac from single clones of HNF-1β knock-out, PAX8 knock-out or control (nt) clones of 786-0 cells at EPAS1 -enhancer E2. The ATAC KIRC element 10767 harboring binding sites for HNF-1β and PAX8 is highlighted in blue, the HIF-1β binding site at KIRC_10770 is marked in orange. f Schematic depiction of transcription factor binding sites for HNF-1β, PAX8 and HIF-1β at EPAS1- enhancer E2. Positions of sgRNAs used for knock-out of the respective binding sites are indicated in orange. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560 . g Expression qPCR analysis for HIF-2α mRNA in pools of 786-0 cells transfected with sgRNA targeting the HNF-1β (E2_HNF-1β) or PAX8 (E2_PAX8) binding site at the EPAS1- enhancer E2. Cells treated with non-targeting sgRNA served as controls. Values are mean + SD. p -values were determined by a two tailed one sample t-test with a hypothetical value of 1. n = 5 (E2_HNF-1β) or n = 6 (E2_PAX8) independent experiments. h Immunoblot analyses for HIF-2α and β-actin in 786-0 cells with intact (nt) or mutated binding sites for HNF-1β (E2_HNF-1β) or PAX8 (E2_PAX8) at the EPAS1 -enhancer E2. Representative blot from two independent experiments with similar results.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: ChIP-sequencing, Binding Assay, Expressing, Two Tailed Test, Western Blot, CRISPR, Activity Assay, Marker, Clone Assay, Knock-Out, Control, Transfection

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Schematic of the two HIF-binding EPAS1 -enhancers E1 and E2 at the PRKCE-EPAS1 locus. The location of HIF-binding motifs and positions of sgRNAs within the KIRC elements are indicated. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560 . b Expression qPCR for HIF-2α mRNA in lysates from primary ccRCC cells isolated from 4 different tumor nephrectomies transfected with a pool of sgRNAs directed against the HIF-binding-sites at EPAS1 -enhancer 1 (E1) and EPAS1 -enhancer 2 (E2_HIF) or a control sgRNA (nt). Values are mean + SD from 4 independent experiments. Significance was determined by a two tailed one sample t-test with a hypothetical value of 1. Expression qPCR for HIF-2α mRNA in lysates from 786-0 ( c ) or RCC4 ( d ) cells transfected with sgRNAs targeting EPAS1 -enhancer 1 (E1), the HIF-binding site of EPAS1 -enhancer 2 (E2_HIF) or both enhancers (E1 + E2_HIF). nt = non-targeting control sgRNA. Values are mean + SD from independent experiments (786-0: n = 7 for nt; n = 6 for E1 and E2_HIF; n = 5 for E1 + E2_HIF; RCC4: n = 4 for nt and E1 + E2 HIF; n = 3 for E1 and E2_HIF). Significance was tested by a two tailed one sample t-test with a hypothetical value of 1. Immunoblot analyses for HIF-2α and β-actin protein in lysates from 786-0 ( e ) or RCC4 ( f ) cells transfected with sgRNAs targeting the enhancers as indicated. Representative blot from three (786-0) or two (RCC4) independent experiments with similar results. Gene set enrichment analysis for differentially expressed genes comparing RNA-seq data generated in 786-0 ( g ) or RCC4 ( h ) cells transfected with sgRNA targeting E2_HIF or non-targeting control guides (nt). One RNA sample of each condition was sequenced in technical duplicates. i Comparison of log 2 (fold change) expression values of genes significantly regulated upon E2_HIF knock-out in 786-0 or RCC4 cells. HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. One RNA sample of each condition was sequenced in technical duplicates.

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Binding Assay, Expressing, Isolation, Transfection, Control, Two Tailed Test, Western Blot, RNA Sequencing, Generated, Comparison, Knock-Out

Journal: Nature Communications

Article Title: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma

doi: 10.1038/s41467-026-68576-0

Figure Lengend Snippet: a Representative immunoblot ( n = 3) for HIF-2α and β-actin protein in lysates from single clones of EPAS1 -enhancer 2 defective (E2_HIF) or control (non-targeting, nt) 786-0 cells. b Xenograft tumor assay in NOD/SCID-gamma mice using EPAS1 -enhancer 2 defective (E2_HIF ko) or intact (nt) clones of 786-0 cells. Volume values are mean ± SD from 6 tumors per cell clone. Two-way ANOVA followed by Bonferroni’s post hoc test comparing tumor volume between mean nt and E2_HIF ko at the indicated time points. Significant differences (** p < 0.01) were detected for comparisons between mean values of E2_HIF ko (2.1 or 2.2) with nt (0.1 or 0.2) xenografts for weeks 8, 9 and 10. c Weight of the explanted xenograft tumors. Data shown are median and interquartile range from 5 (E2_HIF 2.1) or 6 (nt 0.1, nt 0.2, E2_HIF2.2) tumors. One-way ANOVA followed by Bonferroni’s post hoc test. d Volcano plot from RNA-seq experiments in E2_HIF ko versus nt clones of 786-0 cells. HIF-2α mRNA as well as HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. Data is from the two clones of cells per condition from a) and b). e GSEA for HIF-binding sites in 786-0 cells and RNA-seq data ( n = 2 clones) from d). DEG: differentially expressed genes, ES: enrichment score. p -value was estimated using adaptive multilevel Monte Carlo scheme and adjusted for multiple testing with the Benjamini–Hochberg method. f CUT&Tag-seq (C&T) for HIF-1β and H3K27ac as well as ATAC-seq at EPAS1 -enhancer 2 and the control locus EGLN3 from E2_HIF ko versus nt clones of 786-0 cells. g CUT&Tag-seq signals for HIF-1β or H3K27ac integrated over 543 ChIP-seq pre-defined HIF1-β binding sites in E2_HIF ko versus nt clones of 786-0 cells. Non-HIF binding enhancers served as control. Signals were centered on the H3K27ac peak within the enhancers. h Comparison of log 2 (fold change) expression values of genes significantly regulated in E2_HIF ko 786-0 cells ( n = 2 clones) or through HIF-2 inhibition by PT2385 in 786-0 cells . HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. i The transcriptional circuitry of HIF-2α expression at the EPAS1 gene locus. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560 .

Article Snippet: Rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals, Ann Arbor, MI, USA; dilution: 1:1,000) and monoclonal mouse anti-HIF-1α antibody (610959, BD Biosciences, New Jersey, USA; dilution: 1:500), goat polyclonal anti-HIF-2α antibody (AF2997, R&D Systems, Minneapolis, USA; dilution: 1:250) and rabbit monoclonal anti-HIF-2α antibody (A700-003, Bethyl, Waltham, USA; dilution: 1:1,000) as well as rabbit polyclonal anti-HIF-1β antibody (NB100-110, Novus Biologicals, Biotechne, Wiesbaden, Germany; dilution: 1:1,000), monoclonal rat anti-hemagglutinin (HA) antibody (11867423001, Roche Diagnostics GmbH, Mannheim, Germany; dilution: 1:1,000) and polyclonal rabbit anti-VHL antibody (68547, Cell Signaling Technology, Danvers, MA, USA; 1:1,000) were applied for protein detection as applicable.

Techniques: Western Blot, Clone Assay, Control, RNA Sequencing, Binding Assay, ChIP-sequencing, Comparison, Expressing, Inhibition

Journal: The Journal of comparative neurology

Article Title: Immunocytochemical Organization and Sour Taste Activation in the Rostral Nuc. Solitary Tract of Mice

doi: 10.1002/cne.24059

Figure Lengend Snippet: Age and Sex of All Animals Used

Article Snippet: Rabbit polyclonal anti-P2X2 (AB_2040054) , synthetic peptide to rat C’ terminus (SQQDSTSTDPKGLAQL) , Alomone Labs, LTD Jerusaleum (APR-003) , 1:500.

Techniques:

Journal: The Journal of comparative neurology

Article Title: Immunocytochemical Organization and Sour Taste Activation in the Rostral Nuc. Solitary Tract of Mice

doi: 10.1002/cne.24059

Figure Lengend Snippet: Primary Antibodies

Article Snippet: Rabbit polyclonal anti-P2X2 (AB_2040054) , synthetic peptide to rat C’ terminus (SQQDSTSTDPKGLAQL) , Alomone Labs, LTD Jerusaleum (APR-003) , 1:500.

Techniques: Sequencing

Journal: The Journal of comparative neurology

Article Title: Immunocytochemical Organization and Sour Taste Activation in the Rostral Nuc. Solitary Tract of Mice

doi: 10.1002/cne.24059

Figure Lengend Snippet: See supplemental Fig. 1 (S1) for P2X2/ChAT immunoreactivity across all nine nTS levels. For each nTS level, a pair of images is shown, Left: photographs of P2X2 (green), ChAT (magenta), and a Nissl background stain (cyan), with nTS boundaries indicated as white dotted lines; Right: computationally extracted chartings of the image for each pixel greater than 2 S.D. of background staining. r1: In rostral sections, P2X2 immunoreactive fibers (green) label the facial nerve root as it passes medially through the dorsal medulla, terminating within the dorsocentral subnucleus of the nTS (r1 & r3). At more caudal levels (i2 – i4), P2X2 immunoreactive fibers expand medially and ventrally to terminate within the medial two-thirds of the nTS. CHAT- GFP primarily labels brainstem motor nuclei and nerve roots throughout the medulla (including DMX, 12n).

Article Snippet: Rabbit polyclonal anti-P2X2 (AB_2040054) , synthetic peptide to rat C’ terminus (SQQDSTSTDPKGLAQL) , Alomone Labs, LTD Jerusaleum (APR-003) , 1:500.

Techniques: Staining

Journal: The Journal of comparative neurology

Article Title: Immunocytochemical Organization and Sour Taste Activation in the Rostral Nuc. Solitary Tract of Mice

doi: 10.1002/cne.24059

Figure Lengend Snippet: See supplemental Fig. 2 (S2) for P2X2/NPY immunoreactivity across all nine nTS levels. In rostral nTS, NPY-immunoreactive fibers are distributed in the medial part of the nTS and within the DMX in areas that also contain ChAT-positive cells and processes. In intermediate nTS, NPY staining is densest lining the 4th ventricle (4V), but also occurs within the area postrema (AP). The nTS boundaries and organization of the figure is as described for Fig. 3.

Article Snippet: Rabbit polyclonal anti-P2X2 (AB_2040054) , synthetic peptide to rat C’ terminus (SQQDSTSTDPKGLAQL) , Alomone Labs, LTD Jerusaleum (APR-003) , 1:500.

Techniques: Staining

Journal: The Journal of comparative neurology

Article Title: Immunocytochemical Organization and Sour Taste Activation in the Rostral Nuc. Solitary Tract of Mice

doi: 10.1002/cne.24059

Figure Lengend Snippet: See supplemental Fig 3. (S3) for P2X2/galanin immunoreactivity across all nine nTS levels. Galanin+ fibers are distributed in a roughly similar pattern to that of NPY, although the peptides do not appear to co-localize within the majority of individual fibers (See Fig. 7). The nTS boundaries and organization of the figure is as described for Fig. 3.

Article Snippet: Rabbit polyclonal anti-P2X2 (AB_2040054) , synthetic peptide to rat C’ terminus (SQQDSTSTDPKGLAQL) , Alomone Labs, LTD Jerusaleum (APR-003) , 1:500.

Techniques:

Journal: The Journal of comparative neurology

Article Title: Immunocytochemical Organization and Sour Taste Activation in the Rostral Nuc. Solitary Tract of Mice

doi: 10.1002/cne.24059

Figure Lengend Snippet: See supplemental Fig. 4 (S4) for P2X2/CGRP immunoreactivity across all nine nTS levels. For each nTS level, a pair of images is shown, Left: photographs of P2X2 (green), CGRP (magenta), and a Nissl background stain (cyan), with nTS boundaries indicated as white dotted lines; Right: computationally extracted charting of the image for each pixel greater than 2 S.D. of background staining. Patterns of P2X2 immunoreactivity are similar to that described in Fig. 3 above. CGRP-immunoreactive fibers appear both within and immediately lateral to the RL subdivision of the nTS. However, a substantial number of CGRP+ fibers also terminate within the RC subdivision of nTS, overlapping the P2X2 terminal field. Moreover, a unique CGRP+ zone occurs laterally beyond the nominal boundaries of the nTS and occupies the DMSp5.

Article Snippet: Rabbit polyclonal anti-P2X2 (AB_2040054) , synthetic peptide to rat C’ terminus (SQQDSTSTDPKGLAQL) , Alomone Labs, LTD Jerusaleum (APR-003) , 1:500.

Techniques: Staining

Journal: Neuroscience Bulletin

Article Title: Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells

doi: 10.1007/s12264-019-00353-0

Figure Lengend Snippet: Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre+/–, and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, GluN2A, and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. *P < 0.05, ***P < 0.001 vs control.

Article Snippet: After blocking in 5% non-fat milk at room temperature for 2 h, the membranes were incubated overnight at 4°C with the following primary antibodies: polyclonal mouse anti-GluN1 (1:1000; BD Pharmingen, Franklin Lakes, NJ), polyclonal rabbit anti-GluN2A (1:200; Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-GluN2B (1:200; Alomone Labs), polyclonal rabbit anti-GluA1 (1:200; Alomone Labs), monoclonal rabbit anti-mGluR1 (1:1000; Cell Signaling Technology, Danvers, MA), monoclonal rabbit anti-mGluR5 (1:500; Abcam), polyclonal rabbit anti-glycine receptor alpha1+alpha2 (1:1000; Abcam), and polyclonal rabbit anti-GABA A receptor alpha1 (GABRA1) (1:1000; Abcam).

Techniques: Western Blot, Expressing