Anti hipk2

Western blot analyses were conducted as previously described [19 (link), 20 (link)]. Briefly, HCT-116 cells were treated by VB (25, 50, and 100 μM) for 48 h, before being lysed and total protein was extracted. Protein samples were separated with 10%SDS-PAGE gel, transferred to a PVDF membrane with a Trans-Blot (Bio-Rad). The membrane was probed with primary antibodies (1: 1000 of anti-HIPK2, 1: 1000 of anti-P53, 1: 1000 of anti- p-p53, 1:1000 of anti-Bax, 1: 1000 of anti-Bcl-2, or 1: 4000 of anti-β-actin; Cell Signaling Technology, Danvers, MA, USA). The signal intensities of protein abundance were quantitatively analyzed by Image J.

Proteins from cultured cells were extracted by utilizing the RIPA lysis buffer (Abcam, USA) according to standard protocol. DC Protein Assay Kit (Bio-Rad, USA) was utilized to quantify the protein concentration. Equal protein from each sample was loaded into SDS-polyacrylamide gels and separated through electrophoresis. Later proteins in the gels were transferred to PVDF membranes (Sigma-Aldrich, USA). 3% BSA was used to block the membranes for 30–60 min at room temperature and then specific primary antibodies were added to incubate at 4 °C overnight. The antibodies were discarded and TBST was utilized to wash the membranes three times before incubation with specific goat anti-rabbit (cat. no. 7074) or goat anti-mouse (cat. no. 7076) horseradish peroxidase-conjugated secondary antibodies (1:3000; Cell Signaling Technology, USA) for 1–2 h at room temperature. Protein band intensities were detected by using the ECL Kit (Bio-Rad). Primary antibodies used in the study were: Anti-E2F1 (1:1500; cat. no. 3742, Cell Signaling Technology, USA); Anti-HIPK2 (1:1500; cat. no. ab221980, Abcam, USA); Anti-HIF1α (1:2500; cat. no. ab179483, Abcam, USA); Anti-β-actin (1: 5000; cat. no. ab8226, Abcam, USA).

Western blotting analysis was performed following a previously described method 20 (link). Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, #R0278) containing protease inhibitor (Sigma-Aldrich, P8340). Equal amounts of total proteins were loaded and resolved by SDS-PAGE gels, followed by transferring to a PVDF membrane, blocking with 5% milk, and probing with primary antibodies. The following primary antibodies were used: anti-CtBP1 (BD Bioscience, USA, #612042), anti-CtBP1 (phospho Ser422) (GeneTex, USA, #GTX55356), anti-CtBP2 (BD Bioscience, #612044), anti-HIPK2 (Cell Signaling, USA, #5091S), anti-BIM (Abcam, China, #ab170589), anti-BIK (Abcam, #ab52182), anti-BAX (Abcam, #ab3191), anti-NOXA (Abcam, #114C307), anti-CASP3 (Sigma-Aldrich, #C9598), anti-CASP7 (Sigma-Aldrich, #C1104), anti-CASP9 (Abcam, #ab184786), anti-p300 (Sigma-Aldrich, #P2859), anti-FOXO3a (Sigma-Aldrich, #V38041), and anti-GAPDH (Thermo Fisher Scientific, #MA5-15738-BTIN). The protein signals were visualized using an ECL detection kit (Sigma-Aldrich, #GERPN2109).