cpsf2 wb 17739 1 ap proteintech cpsf3 wb 11609 1 ap proteintech cpsf4 wb a301 584a bethyl  (Proteintech)

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: Splicing factors are transcriptional targets for E2F1 and MYCN in neuroblastoma cell lines. (A) The mRNA expression level of splicing factors CPSF3 , SRSF1 and HNRNPA3 in CHP‐134 and GI‐ME‐N cell lines. Significance was calculated with an unpaired t ‐test. Results are the mean values ± SD; n = 3 independent experiments (each with three technical replicates). Immunoblot showing the protein expression levels of MYCN and E2F1 in the CHP‐134 and GI‐ME‐N cell lines. GAPDH served as a loading control for this experiment. (B, C) ChIP assays denoting the binding affinity of MYCN and the E2F1 transcription factors on the promoter region of the splicing factor genes CPSF3 , HNRNPA3 and SRSF1 in both CHP‐134 and GI‐ME‐N cell lines. Results represent mean percentage enrichment values ± SD; Significance was calculated with one‐way ANOVA with Tukey's multiple comparison test; ( n = 3 independent experiments, each with three technical replicates). PARP2 and CDC6 served as a positive control for the binding of MYCN and E2F1 respectively. (D) The mRNA expression levels of splicing factors CPSF3 , HNRNPA3 and SRSF3 in a MYCN SHEP‐21N Tet‐off inducible cell line treated with (+DOX) or without (−DOX) doxycycline; Significance was calculated with an unpaired t ‐test. Results represent mean expression values ± SD; n = 3 independent experiments (each with three technical replicates). A Representative immunoblot ( n = 3 independent experiments) showing the protein expression levels of MYCN and E2F1 in SHEP‐21N (−DOX) MYCN overexpressing cells and the SHEP‐21N (+DOX) non‐MYCN‐expressing cells following treatment with doxycycline for 72 h. GAPDH served as a loading control for this experiment. (E) Chromatin immunoprecipitation (ChIP) assays denoting the binding affinity of the MYCN transcription factor on the promoter region of the splicing factor genes CPSF3 , HNRNPA3 and SRSF3 in both SHEP‐21N cells overexpressing MYCN (−DOX) and in cells with decreased MYCN expression (+DOX for 72 h). Results represent mean percentage enrichment values ± SD; Significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). PARP2 served as a positive control for the binding of MYCN. (F) Chromatin immunoprecipitation (ChIP) assays denoting the binding affinity of the E2F1 transcription factor on the promoter region of the splicing factor genes CPSF3 , HNRNPA3 and SRSF3 in both SHEP‐21N cells overexpressing MYCN (−DOX) and in cells with decreased MYCN expression (+DOX for 72 h). Results represent mean percentage enrichment values ± SD; Significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). CDC6 served as a positive control for the binding of E2F1.

Article Snippet: The following antibodies were used in immunoblots: β‐actin (3700S, Cell Signaling Technology, Danvers, MA, USA, dilution 1 : 2000), E2F1 (3742S, Cell Signaling Technology, dilution 1 : 1000), symmetric dimethyl arginine (SDMe) (13222S, Cell Signaling Technology, dilution 1 : 1000), FLAG (clone M2, F1804, Sigma, St. Louis, MO, USA, 1 : 1000), GAPDH (clone 6C5, MAB374, Millipore, Burlington, MA, USA, 1 : 2000), Smac/DIABLO (2954S, Cell Signaling Technology, dilution 1 : 1000), BIM (BCL2L11) (2819, Cell Signaling Technology, dilution 1 : 1000), ACIN1 (A300‐999A, Bethyl, Montgomery, TX, USA, dilution 1 : 1000) CFLAR (AV00022‐QC0240, Sigma‐Aldrich, dilution 1 : 1000), MYCN (SC‐53993, Santa Cruz, Dallas, TX, USA, dilution 1 : 1000), PRMT5 (79998S, Cell Signaling Technology, dilution 1 : 1000), CPSF3 (SC‐393001, Santa Cruz, dilution 1 : 1000), CPSF4 (15023‐I‐AP, ProteinTech, Rosemont, IL, USA, dilution 1 : 1000), SRSF1 (SC‐33652, Santa Cruz, dilution 1 : 1000), SRSF3 (51039S, Cell Signaling Technology, dilution 1 : 1000), SRSF4 (H303‐670A, Bethyl, dilution 1 : 1000), HNRNPA3 (25142‐I‐AP, ProteinTech, dilution 1 : 1000), HNRNPM (TA803154, ORIGENE, Rockville, MD, USA, dilution 1 : 1000).

Techniques: Expressing, Western Blot, Control, Binding Assay, Comparison, Positive Control, Chromatin Immunoprecipitation

Journal: Cell reports

Article Title: Non-canonical isoforms of the mRNA polyadenylation factor WDR33 regulate STING-mediated immune responses

doi: 10.1016/j.celrep.2024.113886

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-CPSF3 , Santa Cruz Biotechnology , Cat# sc-393001.

Techniques: Virus, Recombinant, Negative Control, Software

Journal: Cell discovery

Article Title: RBBP6 maintains glioblastoma stem cells through CPSF3-dependent alternative polyadenylation.

doi: 10.1038/s41421-024-00654-3

Figure Lengend Snippet: Fig. 7 Working model of RBBP6 in regulating GSCs. RBBP6 regulates the maintenance of GSCs. Depletion of RBBP6 leads to the elimination of K63-linked ubiquitination of CPSF3, resulting in the destabilization of CPSF3 and the subsequent shortening of the 3’ UTRs of MYC ceRNAs. This shortening promotes the release of miR-590-3p from the truncated UTRs, thereby reducing MYC expression and hindering the survival and progression of GSCs.

Article Snippet: The antibodies used in this study wereRBBP6 (Bethyl Laboratories, Cat# A304-975A), CPSF3 (Proteintech, Cat# 11609-1-AP), CPSF2 (Proteintech, Cat# 17739-1-AP), NUDT21 (Proteintech, Cat# 10322-1-AP), CSTF2 (Proteintech, Cat# 26825-1-AP), HA-tag (Cell signaling Technology, Cat# 3724 S), Flag-tag (Sigma, Cat # F1804), MYC-tag (Proteintech, Cat # 16286- 1-AP), MYC (Cell signaling Technology, Cat# D3N8F), GAPDH (Proteintech, Cat# 60004-1-Ig).

Techniques: Ubiquitin Proteomics, Expressing