2.6. Western blot

Total protein was extracted from cells and tissue with the addition of 20 mM N‐ethylmaleimide (Sigma‐Aldrich), and protein was quantified with a BCA kit. Protein samples were separated on 9% SDS‐PAGE gels and transferred to nitrocellulose‐filter membranes. Then, membranes were probed with the following primary antibodies against RNF4 (1:500; Creative Diagnostics, USA), GAPDH (1:10000; ABclonal, USA), actin (1:1000; Proteintech, USA), ubiquitin (1:600; Santa Cruz Biotechnology, USA), PML (1:1000; MBL, Japan), SUMO‐1 (1:200; Santa Cruz Biotechnology, USA), SUMO‐2/3 (1:500; Abcam, USA), p53 (1:800; Abcam, USA) and p‐p53 (1:1000; Cell Signaling Technology, USA). Immunoblots were observed by a LI‐COR Imaging System (LI‐COR Biosciences, Lincoln, NE), and Odyssey software was used to analyse band intensities (area × OD), which were normalized to GAPDH/Actin. Results are reported as fold changes normalized to control values.