Ecl stable peroxide solution

The proteins in the lysates were resuspended using SDS‐PAGE electrophoresis and transferred to a nitrocellulose membrane, which was then blocked with PBS/Tween‐20 containing 5% nonfat milk. The membrane was incubated with antibodies against REV7 (Abcam), PRDX2 (Abnova), GAPDH (Beyotime), Lamin B1 (Santa Cruz, CA, USA), Bcl‐2 and BAX (Cell Signaling Technology, Danvers, MA, USA). The protein‐bound antibodies were detected using an enhanced chemiluminescence (ECL) stable peroxide solution (Beyotime). All protein bands were visualized using a FluoroChem MI imaging system (AlphaInnotech, Santa Clara, CA, USA) at room temperature.

The cells were lysed in lysis buffer (Promega Corporation) and centrifuged at 4˚C, 12,000 x g for 10 min. The supernatant was collected and subjected to western blotting. Protein concentration was subsequently measured using a BCA Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Protein (50 µg) from each lysate was fractionated by 10% SDS-PAGE. The samples were electrophoresed for 2 h and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). After being blocked with 5% BSA in TBS-0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were blotted with HuR (Abcam; cat. no. ab220342) or α-Tubulin (Beyotime Institute of Biotechnology; cat. no. AF0001) primary antibodies at 1:1,000 dilutions. The membranes were then incubated with the appropriate horseradish peroxidase-coupled secondary antibody (Beyotime Institute of Biotechnology; cat. no. A0277) at a 1:2,000 dilution for 1 h at room temperature. After the membranes were washed with TBST, the blots were incubated with enhanced chemiluminescence (ECL) stable peroxide solution (Beyotime Institute of Biotechnology). All blots were visualized using a FluoroChem MI imaging system (Alpha Innotech Corporation) at room temperature. Densitometry was performed using ImageJ v1.8.0-172 software (National Institutes of Health).