anti-prdm5 antibody 7d4c12  (Novus Biologicals)

Journal: Nature

Article Title: Necroptosis microenvironment directs lineage commitment in liver cancer

doi: 10.1038/s41586-018-0519-y

Figure Lengend Snippet: a, ATAC-seq density heat map of chromatin regions that are differentially accessible between HCC and ICC. Peaks are ranked according to the fold-change in signal in normalized ATAC fragment counts in ICC versus HCC. The data are expressed as smoothed normalized fragment pseudocounts in 25-base-pair (bp) windows ± 1 kb around the centre of peaks. The lateral bars on the left depict whether the ATAC signal is significantly increased (green) or decreased (red) in ICC compared to HCC as assessed with EdgeR. Tbx3- and Prdm5-associated regulatory elements are indicated. For each transcription factor (TBX3 or PRDM5), n = 4 cases, two-sided moderated t-statistics. b, Heat map of transcriptome data comparing HCC and ICC showing differentially expressed probes. Probes matching Tbx3 and Prdm5 are indicated by arrows. For each transcription factor (TBX3 or PRDM5), n = 4 cases, two-sided moderated t-statistics. c, d, Integrative analysis of chromatin accessibility (left) and transcriptome data (right) around Tbx3 (c) and Prdm5 (d) genes comparing HCC and ICC. Chromatin accessibility is expressed as smoothed, normalized fragment pseudo-counts in 100-bp windows. Absolute gene expression is represented in log scale. e, f, Gene expression in 199 human HCC and ICC of TBX3 (e) and PRDM5 (f). ****P < 0.0001, Student’s two-sided t-test. Data are mean ± s.d. g, Immunohistochemistry staining for K19 and HNF4α of HDTV-derived tumours after co- delivery of pCaMIN and a Prdm5-overexpression transposon (Prdm5 OE) (n = 4), pCaMIN plus Tbx3 shRNA (shTbx3) (n = 5), or pCaMIN plus shTbx3 and Prdm5 OE (n = 3) in p19Arf−/− mice. Scale bars, 100 μm. h, Schematic representation of the proposed model. DAMPs, damage-associated molecular patterns.

Article Snippet: Immunoprecipitations for TBX3 with HCC H1 and H4 chromatin were performed with an anti-TBX3 antibody (Santa Cruz, sc-17871, 5 μg), and the immunoprecipitations for PRDM5 with ICC E9 and E10 chromatin were performed with anti-PRDM5 antibody (Millipore, MABE972, 5 μg).

Techniques: Expressing, Immunohistochemistry, Staining, Derivative Assay, Over Expression, shRNA

Journal: Nature

Article Title: Necroptosis microenvironment directs lineage commitment in liver cancer

doi: 10.1038/s41586-018-0519-y

Figure Lengend Snippet: a, b, ChIP–seq density heat map for two biological replicates in the global set of reproducible peaks detected for Tbx3 (a) and Prdm5 (a) following the irreproducible discovery rate workflow (a and b, left) and corresponding ATAC-seq signal (a and b, right). Peaks are ranked according to the average ChIP–seq signal across replicates. The data are expressed as normalized reads per million mapped reads (RPM). The signal is shown 5 kb upstream and downstream of the centre of the ChIP–seq peaks. c, d, Heat maps depicting gene expression changes after Tbx3 shRNA-mediated (c) and Prdm5 shRNA-mediated (d) suppression. Only direct Tbx3 and Prdm5 targets are shown. Data are expressed as z-score. For each transcription factor (TBX3 or PRDM5), n = 4 cases (2 shRNAs per target, biological duplicates for each) and n = 2 controls (1 control shRNA in duplicate), two-sided moderated t-statistics. e, f, Heat maps depicting gene expression changes after Tbx3 (e) and Prdm5 (f) shRNA-mediated stable knockdown. Each knockdown experiment was performed in established cell lines from two different clones using two different shRNAs. In these heat maps, both direct and indirect Tbx3 and Prdm5 ChIP–seq-derived gene targets are shown. Differentially regulated genes were separated into direct or indirect Tbx3 or Prdm5 targets based on the presence or absence of proximal ChIP–seq peaks (<100 kb from the TSS or inside the gene body of deregulated genes). Data are expressed as row Z-score. For each transcription factor (TBX3 or PRDM5), n = 4 cases (2 shRNAs per target, biological duplicates for each) and n = 2 controls (1 control shRNA in duplicate), two-sided moderated t-statistics. g, Functional over-representation map depicting MSigDB canonical pathways associated to all/direct target/indirect target genes perturbed after Tbx3 and Prdm5 knockdown. The size of dots is proportional to the P value based on the hypergeometric distribution obtained when testing for over-representation, and their colour denotes whether the term is enriched for up or downregulated gene list. These data show regulation of distinct downstream pathways between Tbx3 (for example, biological oxidation, developmental biology) and Prdm5 (for example, extracellular matrix organization, collagen formation or Erbb signalling) (n = 4 cases; 2 shRNAs per target, biological duplicates for each, and n = 2 controls; 1 control shRNA in duplicate). h, qRT–PCR analysis of epigenetic modifiers from livers 3 days after Epo or HDTV treatment. All significantly regulated genes are shown (n = 3). P values determined by Student’s two-sided t-test. Data are fold changes of the mean.

Article Snippet: Immunoprecipitations for TBX3 with HCC H1 and H4 chromatin were performed with an anti-TBX3 antibody (Santa Cruz, sc-17871, 5 μg), and the immunoprecipitations for PRDM5 with ICC E9 and E10 chromatin were performed with anti-PRDM5 antibody (Millipore, MABE972, 5 μg).

Techniques: ChIP-sequencing, Expressing, shRNA, Clone Assay, Derivative Assay, Functional Assay, Quantitative RT-PCR

Journal: Nature

Article Title: Necroptosis microenvironment directs lineage commitment in liver cancer

doi: 10.1038/s41586-018-0519-y

Figure Lengend Snippet: a, Immunocytochemistry of isolated single cell lines of HDTV-derived HCC and Epo-derived ICC tumours. Depicted are representative co-staining images of K19 (red) and DAPI (blue). Scale bars, 100 μm. Experiment was performed twice with similar results. b, Schematic outline of the generation of clonal cell lines of Epo and HDTV tumours for subcutaneous injection into immunodeficient Rag2−/− mice. c, Representative micrographs of sections from subcutaneously grown HCC (see b; top) and ICC (bottom) with H&E (left) and K19 (right) staining. These data show that both HCC and ICC phenotypes are stably maintained even after in vitro passaging and in vivo retransplantation procedures in mice (n = 3). Scale bars, 100 μm. d, Bi-clustering of pairwise Pearson’s correlations based on normalized ATAC-seq fragment pseudo-counts for differentially accessible areas in ICC (n = 4 single cell clones) and HCC (n = 4 single cell clones). e, f, qRT–PCR analysis for Tbx3 (e) or Prdm5 (f) in mouse HCC or ICC cells (n = 4 single cell clones each). ***P = 0.0004, ****P < 0.0001, Student’s two-sided t-test. Data are mean ± s.d.

Article Snippet: Immunoprecipitations for TBX3 with HCC H1 and H4 chromatin were performed with an anti-TBX3 antibody (Santa Cruz, sc-17871, 5 μg), and the immunoprecipitations for PRDM5 with ICC E9 and E10 chromatin were performed with anti-PRDM5 antibody (Millipore, MABE972, 5 μg).

Techniques: Derivative Assay, Immunocytochemistry, Isolation, Staining, Injection, Stable Transfection, In Vitro, Passaging, In Vivo, Clone Assay, Quantitative RT-PCR

Journal: Nature

Article Title: Necroptosis microenvironment directs lineage commitment in liver cancer

doi: 10.1038/s41586-018-0519-y

Figure Lengend Snippet: a, Representative micrographs of immunostaining for HNF4α or K19 on tumour sections after Epo delivery of pCaMIN transposon vector co-expressing control shRNA (shRen) and full-length Tbx3 (pCAMINshRen + Tbx3 Epo) or pCaMIN vector co-expressing Prdm5 shRNA and full-length Tbx3 (pCAMINPrdm5_1 + Tbx3 Epo) (n = 3). Scale bars, 100 μm. b, Representative micrograph of tumours induced by Epo delivery of pCaMIN and Tbx3 overexpression in ROSAmT/mG × Alb-cre × p19Arf−/− mice showing DAPI (blue) and mGFP (green) positivity (n = 6). Scale bar, 100 μm. c, qRT–PCR analysis for Tbx3 in mouse HCC cells stably expressing shRNAs targeting Tbx3 (shTbx3_1 and shTbx_2; n = 3). Data are mean ± s.d. d, qRT–PCR analysis for Prdm5 in mouse ICC cells stably expressing shRNAs targeting Prdm5 (shPrdm5_1 and shPrdm5_2; n = 2). Data are mean ± s.d.

Article Snippet: Immunoprecipitations for TBX3 with HCC H1 and H4 chromatin were performed with an anti-TBX3 antibody (Santa Cruz, sc-17871, 5 μg), and the immunoprecipitations for PRDM5 with ICC E9 and E10 chromatin were performed with anti-PRDM5 antibody (Millipore, MABE972, 5 μg).

Techniques: Immunostaining, Plasmid Preparation, Expressing, shRNA, Over Expression, Quantitative RT-PCR, Stable Transfection

Journal: Journal of Cancer

Article Title: Downregulation of promoter methylation gene PRDM5 contributes to the development of tumor proliferation and predicts poor prognosis in gastric cancer

doi: 10.7150/jca.59998

Figure Lengend Snippet: PRDM5 mRNA expression and protein expression are downregulated in GC. A-B Expression of PRDM5 was frequently downregulated in gastric tumor tissues (tumor) compared with adjacent or normal gastric tissue samples (normal) in public databases. C-D mRNA levels of PRDM5 detected by Q-rtPCR and protein levels of PRDM5 detected by western blot in tumors and adjacent tumors. Data are shown as mean ± SD * p < 0.05;** p < 0.01; *** p < 0.001, **** p < 0.0001.

Article Snippet: In the immunostaining of the tissue microarray (TMA), the primary antibody was anti-PRDM5 antibody (1:300 dilution, number: 7D4C12, NOVUS).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer

Article Title: Downregulation of promoter methylation gene PRDM5 contributes to the development of tumor proliferation and predicts poor prognosis in gastric cancer

doi: 10.7150/jca.59998

Figure Lengend Snippet: Univariate and multivariate Cox proportional hazard analyses for cancer-specific survival

Article Snippet: In the immunostaining of the tissue microarray (TMA), the primary antibody was anti-PRDM5 antibody (1:300 dilution, number: 7D4C12, NOVUS).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Downregulation of promoter methylation gene PRDM5 contributes to the development of tumor proliferation and predicts poor prognosis in gastric cancer

doi: 10.7150/jca.59998

Figure Lengend Snippet: Correlation of PRDM5 in GC and clinical parameters in 162 cases

Article Snippet: In the immunostaining of the tissue microarray (TMA), the primary antibody was anti-PRDM5 antibody (1:300 dilution, number: 7D4C12, NOVUS).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Downregulation of promoter methylation gene PRDM5 contributes to the development of tumor proliferation and predicts poor prognosis in gastric cancer

doi: 10.7150/jca.59998

Figure Lengend Snippet: PRDM5 inhibited the proliferation and migration of GC cells. A-E. The proliferation ability of GC cells was inhibited by PRDM5. F. The proliferation ability of cells was enhanced after PRDM5 knockdown. G. Statistical results of cell cloning experiments. H. Cell migration assay after changing PRDM5 expression. I. Statistical results of cell migration assay.

Article Snippet: In the immunostaining of the tissue microarray (TMA), the primary antibody was anti-PRDM5 antibody (1:300 dilution, number: 7D4C12, NOVUS).

Techniques: Migration, Knockdown, Cloning, Cell Migration Assay, Expressing

Journal: Journal of Cancer

Article Title: Downregulation of promoter methylation gene PRDM5 contributes to the development of tumor proliferation and predicts poor prognosis in gastric cancer

doi: 10.7150/jca.59998

Figure Lengend Snippet: The promoter of PRDM5 is often methylated. A. The methylated specific primers and unmethylated specific primers were used to detect the difference of methylation between tumor and normal tissues. M primers specific to methylated template DNA, U primers specific to unmethylated template DNA, T tumor, N normal. B. PRDM5 protein expression in cells treated with methylation inhibitors at different concentrations (µM). C-D. Proliferation ability of AGS( C ) and SGC-7901( D ) cells which were treated with methylation inhibitors at appropriate concentrations. E. MEXPRESS analysis about the correlation between DNA methylation of CpG islands in the 5′ promoter region and expression level of PRDM5.

Article Snippet: In the immunostaining of the tissue microarray (TMA), the primary antibody was anti-PRDM5 antibody (1:300 dilution, number: 7D4C12, NOVUS).

Techniques: Methylation, Expressing, DNA Methylation Assay

Journal: Nature Communications

Article Title: Endocannabinoid signaling regulates the reinforcing and psychostimulant effects of ketamine in mice

doi: 10.1038/s41467-020-19780-z

Figure Lengend Snippet: a , b Repeated ketamine exposure (15 mg/kg) increased PRDM5 protein expression in the nucleus of DLS neurons (unpaired two-tailed t test, t (6) = 2.533, P = 0.0445). PRDM5 is observed as two bands at 66 and 70 kDa (two isoforms). c Co-immunostaining for PRDM5 (red), NeuN (green), GFAP (wathet blue), and DAPI (deep blue) in the DLS. d Ketamine (15 mg/kg) increased PRDM5 protein expression in DLS neurons (unpaired two-tailed t test, t (6) = 3.572, P = 0.0118). e , g EMSA experiments were performed with 5 μg of PRDM5 protein and biotin-labeled Mgll DNA oligos (probe1 and probe2). The shifted protein–DNA band (red rectangle; lanes 2, 3, and 6 in e ; lanes 2, 3, and 7 in g was competed with excess and unlabeled DNA oligos (lane 4). Preincubation with anti-PRDM5 antibodies resulted in supershifted antibody–protein–DNA bands (blue rectangle; lanes 7, 8, and 9 in e ; lanes 9 and 10 in g that migrate more slowly in the native gel. f , h Ketamine (15 mg/kg) enriched PRDM5 at the promoter of gene Mgll (site 1 and site 2; site1, unpaired two-tailed t test, t (6) = 12.87, P < 0.0001; site2, unpaired t test, t (6) = 20.32, P < 0.0001). Compared to SAL group, * P < 0.05, *** P < 0.001. i , j shRNA2 targeting Prdm5 effectively interfered with PRDM5 expression (unpaired two-tailed t test, t (10) = 3.908, P = 0.0029). Compared to SCR group, ** P < 0.01. k AAV-sh Prdm5 reduced ketamine-seeking behaviors ( n = 21 mice/group, two-way repeated measured ANOVA, followed by Dunnett’s multiple comparisons test, virus F (3,48) = 38.24, P < 0.0001; time F (8,384) = 2.848, P = 0.0044; interaction, F (24,384) = 2.056, P = 0.0027). l AAV-sh Prdm5 reduced ketamine-taking behaviors ( n = 21 mice/group, two-way repeated measured ANOVA, followed by Dunnett’s multiple comparisons test, virus F (3,48) = 31.97, P < 0.0001; time F (8,384) = 2.554, P = 0.0101; interaction, F (24,384) = 1.016, P = 0.4441). m AAV- shPrdm5 restored the DLS 2-AG level in ketamine self-administration test (one-way ANOVA, followed by Dunnett’s multiple comparisons test, F (3,30) = 5.848, P = 0.0029; SCR + KET vs. SCR + SAL, P = 0.0461; SCR + KET vs. AAV-sh Prdm5 + KET, P = 0.0123). n AAV-sh Prdm5 could not reduce ketamine-induced hyperlocomotor activity ( n = 21 mice/group, two-way repeated measured ANOVA, followed by Dunnett’s multiple comparisons test: virus F (3,80) = 7.706, P = 0.0001; time F (7,560) = 8.764, P < 0.001; interaction, F (21,560) = 2.867, P < 0.001). o AAV- shPrdm5 restored the 2-AG level in the DLS in ketamine hyperlocomotion test (one-way ANOVA, followed by Dunnett’s multiple comparisons test, F (3,28) = 5.727, P = 0.0035; SCR + KET vs. SCR + SAL, P = 0.0142; SCR + KET vs. AAV-sh Prdm5 + KET, P = 0.0031). Compared to SCR + SAL group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared to SCR + KET group, # P < 0.05, ## P < 0.01, ### P < 0.001. Data are shown as mean ± SEM. SAL saline, KET ketamine, SCR scrambled sh RNA. Source data provided as a file.

Article Snippet: The cross-linked chromatin from bilateral punches of DLS (tissues from three mice were pooled together as one sample) was digested into an average of ~150 bp fragment by micrococcus and then was sonicated to release from nuclear, then immunoprecipitated with specific PRDM5 antibody (5 μg, ChIP grade, #sc-376277x, Santa Cruz) or an IgG control.

Techniques: Expressing, Two Tailed Test, Immunostaining, Labeling, Virus, Activity Assay, Saline

Journal: Nature Communications

Article Title: Endocannabinoid signaling regulates the reinforcing and psychostimulant effects of ketamine in mice

doi: 10.1038/s41467-020-19780-z

Figure Lengend Snippet: a , b Ketamine decreased MAGL expression (unpaired two-tailed t test, t (6) = 2.732, P = 0.0341) but increased PRDM5 expression in the cultured SPNs (unpaired two-tailed t test, t (6) = 2.527, P = 0.0449). Ketamine did not affect DAGL expression (unpaired two-tailed t test, t (6) = 0.1882, P = 0.8569). c Ketamine increased the 2-AG level in the cultured SPNs (unpaired two-tailed t test, t (6) = 2.951, P = 0.0256). d Representative immunofluorescent images of PRDM5 (green) and DAPI (blue). e Ketamine increased PRDM5 expression (unpaired two-tailed t test, t (14) = 2.89, P = 0.0119). f , g Representative immunofluorescent images of MAGL (red) and DAPI (blue). Ketamine decreased MAGL expression (unpaired two-tailed t test, t (14) = 7.16, P < 0.0001). Compared to SAL group, * P < 0.05, ** P < 0.01. h , i LV-sh Prdm5 restored MAGL expression downregulated by ketamine (one-way ANOVA, followed by Dunnett’s multiple comparisons test: PRDM5 F (3,8) = 95.58, P < 0.0001; VEH + KET vs. VEH + SAL, P = 0.0071, VEH + KET vs. LV-sh Prdm5 + KET, P < 0.0001; MAGL F (3,8) = 20.09, P = 0.0004; VEH + KET vs. VEH + SAL, P = 0.0299, VEH + KET vs. LV-sh Prdm5 + KET, P = 0.0008). j LV-sh Prdm5 reversed ketamine-elevated 2-AG level (one-way ANOVA, followed by Dunnett’s multiple comparisons test, F (3,12) = 5.395, P = 0.0139; VEH + KET vs. VEH + SAL, P = 0.0386, VEH + KET vs. LV-sh Prdm5 + KET, P = 0.0364). Compared to SCR + SAL group, * P < 0.05; compared to SCR + KET group, # P < 0.05, ### P < 0.001. Data are shown as mean ± SEM. SAL saline, KET ketamine. Source data provided as a file.

Article Snippet: The cross-linked chromatin from bilateral punches of DLS (tissues from three mice were pooled together as one sample) was digested into an average of ~150 bp fragment by micrococcus and then was sonicated to release from nuclear, then immunoprecipitated with specific PRDM5 antibody (5 μg, ChIP grade, #sc-376277x, Santa Cruz) or an IgG control.

Techniques: Expressing, Two Tailed Test, Cell Culture, Saline

Journal: Nature Communications

Article Title: Endocannabinoid signaling regulates the reinforcing and psychostimulant effects of ketamine in mice

doi: 10.1038/s41467-020-19780-z

Figure Lengend Snippet: a Simplified scheme of PRDM5-MAGL-2-AG-CB1R pathway in normal mice. b Simplified scheme of PRDM5-MAGL-2-AG-CB1R pathway in mice under ketamine conditioning paradigm. Ketamine represses Mgll gene transcription by enhancing the binding of PRDM5 at the promoter of gene Mgll , leading to the decreased MGL expression and 2-AG hydrolysis. Consequently, increased 2-AG activates CB1R signaling, resulting in ketamine addiction-related dendritic remodeling and behaviors.

Article Snippet: The cross-linked chromatin from bilateral punches of DLS (tissues from three mice were pooled together as one sample) was digested into an average of ~150 bp fragment by micrococcus and then was sonicated to release from nuclear, then immunoprecipitated with specific PRDM5 antibody (5 μg, ChIP grade, #sc-376277x, Santa Cruz) or an IgG control.

Techniques: Binding Assay, Expressing