Western blotting

After 48 h transfection, cells were lysed by RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China). Protein concentration was detected with bicinchoninic acid (BCA) method. Separated proteins were denatured at 95 °C for 5 min. Equal amount of protein (20 μg) was added into each well in 12% SDSPAGE, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Afterwards, PVDF membranes were blocked in 5% skim milk for 1 h at room temperature, following the incubation with specific primary antibodies at 4 °C overnight. PVDF membranes were then sealed with secondary antibody for 1 h at 37 °C. The protein signals were visualized with ECL solution (Millipore) and scanned by QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). All antibodies used in this study were listed as the following: anti-SPOP (Abcam, ab192233); anti-CHAF1A (Cell signaling Technology, CST#5480); anti-HA (Abcam, ab9110); anti-p62 (Cell signaling Technology, CST#23,214); anti-Beclin-1 (Cell signaling Technology, CST#4122); anti-β-actin (Cell signaling Technology, CST#41,470); anti-FLAG (Cell signaling Technology, CST#14,793).