pum1 rc201219 orf  (OriGene)

Journal: bioRxiv

Article Title: Identification of PIM1 substrates reveals a role for NDRG1 in prostate cancer cellular migration and invasion

doi: 10.1101/2020.01.21.913962

Figure Lengend Snippet: A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates PUM1 (A), CHMP7 (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.

Article Snippet: Constructs utilized are as follows: pCDNA 3.1 (+)-AR-FLAG , NDRG1 (HG14119-CF, Sino Biologicals), CHMP7 (HG14273-CF, Sino Biologicals), PUM1 (RC201219, Origene), KIF18A (Addgene plasmid # 23002), pCDNA 3.1 (+)-WT PIM1 , pCDNA 3.1 (+)-KD (K67M) , pLB(N)CX-WT PIM1 , pLB(N)CX-KD PIM1 . pCDNA 3.1 (+)- AS (L120G) PIM1 and pLB(N)CX-AS PIM1 (L120G) was generated using site-directed mutagenesis.

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Labeling, Magnetic Beads

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Schematic of full-length human SPIN1 and SPIN3 3′UTRs ( A ). PBE-like motifs responsible for PUF-domain binding are in red and UGUA core motifs are in black ( SPIN3 ). Short 3′UTR fragments containing PBE motifs, which were used for luciferase reporter assays, and full-length 3′UTRs are indicated in brackets, with position within the 3′UTR starting from the end of the stop codon. Enrichment of SPIN1 and SPIN3 in RIP-PUM1 and RIP-PUM2 (indicated by RIP P1 and RIP P2, respectively) was measured via RT-qPCR and was compared to the negative control (nonimmune IgG, indicated by RIP IgG) ( B ). Influence of PUM1 and PUM2 proteins on endogenous SPIN mRNA level ( C ). PUM1 and PUM2 siRNA knockdown efficiencies are shown on the left. Total RNA was isolated from TCam-2 cells in the presence of actinomycin D. SPIN expression was measured via RT-qPCR and was compared to that in untransfected cells. PUM1 or PUM2 overexpression downregulated endogenous SPIN1 as measured by western blotting ( D ). Graphs represent average values with standard errors. P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Negative Control, Isolation, Expressing, Over Expression, Western Blot

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: The effects of PUM proteins on SPIN expression were assessed using a dual luciferase assay. Luciferase reporter constructs carrying full-length 3′UTRs for SPIN1 (upper panel) or SPIN3 (lower panel) were tested with PUM1 (P1) or PUM2 (P2) overexpression or empty pCMV6-entry vector (pC) ( A ). Effects of PUM overexpression on luciferase reporter construct carrying full-length GAPDH mRNA 3′UTR, which lacks PBE motifs (negative control) ( B ). Effects of siRNA-mediated PUM1 (P1 KD) or PUM2 (P2 KD) knockdown (KD) on luciferase reporter constructs carrying full-length SPIN 3′UTRs ( C ). Effects of PUM1 or PUM2 overexpression ( D ). or knockdown ( E ). on luciferase constructs containing short SPIN1 or SPIN3 3′UTR fragments. ** P ≤ 0.005, *** P ≤ 0.0005, **** P ≤ 0.00005.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: Expressing, Luciferase, Construct, Over Expression, Plasmid Preparation, Negative Control

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: TCam-2 cells were transfected with constructs encoding PUM1 (P1), PUM2 (P2), or empty vector and cultured for 48 h. Apoptosis was measured as described for SPINs ( A ). Dot-plot showing quality of TCam-2 cell separation into living, necrotic, and early or late apoptotic populations after transfection with empty vector ( B ), PUM1 ( C ), or PUM2 ( D ) constructs.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: Transfection, Construct, Plasmid Preparation, Cell Culture

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: PUM1 and PUM2 were separately overexpressed and the effects on cell cycle progression were measured 72 h later.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: