Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody

Protein samples were loaded on a polyacrylamide gel (50ug/lane) and separated by electrophoresis. Resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with no-fat milk for 2 hours at room temperature. The membranes were probed with affinity-purified anti-TXNRD1 (1:1000; Hangzhou HuaAn Biotechnology Co. Ltd.), anti-TXNL1 (1:1000; Abcam) or anti-TRX1 (1:1000; Abcam) antibody at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:3000; Pierce). To normalize the loaded samples, affinity purified anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:3000; Proteintech) was used, followed by incubation with HRP-conjugated anti-rabbit IgG (1:3000; Pierce). Images were acquired with the ChemiDoc Tm XRS+ imaging system and analyzed with imaging lab software (Bio-RAD). The density of the band of interest proteins (TXNRD1, TXNL1 or TRX1) was measured and normalized to the density of the band of GAPDH.

9human colorectal cancer cell lines (HCT116, LoVo, RKO, Caco2, HT29, SW48, SW480, SW620, and DLD1) used in this study were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The human normal colonic epithelial cell line FHC was purchased from the Wuhan University Type Culture Collection (Wuhan, China). HCT116, LoVo, and SW620 cells were cultured in McCoy's 5A, F12k, and L15 media, respectively, each containing 10% fetal bovine serum (FBS).RKO, Caco2, HT29, SW48, and SW480 cells were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. DLD1 and FHC cells were incubated in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% FBS. All cell lines were incubated at 37 °C in a CO₂ incubator, except for SW620 cells, which were incubated exposed to air.AZD5153、BMN673 and MK1775 were bought from Selleck (Shanghai, China). The anti-c-Myc, anti-cleaved poly(ADP-ribose) polymerase (PARP), anti-cleaved-caspase-3, anti-Wee1,anti-CDK1,anti-phospho-CDK1 (P-CDK1), anti-γ-H2AXand anti-Ki-67 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-BRD4 antibody was from Abcam (Cambridge, MA, USA), and the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from Proteintech Group, Inc. (Wuhan, Hubei, People's Republic of China).