TRIB3 Protein Expression Analysis by Western Blot

Western blot was used to assess TRIB3 protein expression as previously described (18 (link)). Briefly, whole cell lysates were prepared and quantified using RIPA reagent. Protein samples were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated in a blocking solution, followed by incubation with rabbit anti human monoclonal antibody against TRIB3 (1:1,000, ab137526, Abcam, USA), SMARCD3 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 3; 1:1,000, ab171075,Abcam, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000, Abcam, USA) for 18 hours at 4 °C. Membranes were then washed three times with Tris-buffered saline Tween (TBST) and incubated with a goat anti-rabbit IgG-AP secondary antibody (1:5,000 dilution, #HA1019, HuaBio, Hangzhou, China) in room temperature for 1 hour. After washing, the proteins on the membrane were detected by an enzyme labeling method and chemiluminescence in accordance with the manufacturer’s instructions (Omni ECL reagent (EpiZyme, Shanghai, China).