HEK293 cells were transfected with wild or mutant pEGFP-C1-PRDM1 overexpression plasmids. pEGFP-C1 vector was also transfected as the negative control group. At 48 hours after transfection, cells were harvested and lysed in RIPA lysis buffer (Beyotime) with 1% Protease Inhibitor Cocktail (Cell Signaling Technology). The total protein was quantitated using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturerʼs instructions. Total protein (20 μg) of each sample was loaded, separated on an SDS–PAGE gel and transferred to a polyvinylidene fluoride membrane (MilliporeSigma). The membrane was blocked, incubated with GFP antibody (1:10,000 dilution, Abcam) and anti-rabbit secondary antibodies (1:5,000 dilution, Proteintech) and with β-actin antibody (1:5,000 dilution, Proteintech) and anti-mouse secondary antibody (1:5,000 dilution, Proteintech). The membranes were scanned using a Chemidoc MP Imaging System (Bio-Rad). Two independent experiments were carried out.
For the CHX chase assay, CHX (Beyotime) was added to the culture medium 24 hours after transfection at a concentration of 0.01 mM. Upon treating with CHX for 0 hours, 4 hours, 8 hours and 12 hours, cell samples were collected and stored at −80 °C until western blot analysis was performed. For each timepoint, three independent experiments were carried out.
For the CHX chase assay, CHX (Beyotime) was added to the culture medium 24 hours after transfection at a concentration of 0.01 mM. Upon treating with CHX for 0 hours, 4 hours, 8 hours and 12 hours, cell samples were collected and stored at −80 °C until western blot analysis was performed. For each timepoint, three independent experiments were carried out.