Flag tag (flag)

The cultured cells and the human tissues were lysed with the appropriate amount of RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 ×protease inhibitor mixture (Roche) and PMSF (Beyotime, Shanghai, China). The proteins in the supernatants were subjected to SDS-PAGE and immunoblotting as described previously (Hou et al., 2019 (link)). The antibodies used were as follows: anti-PARP (Cell Signaling Technology), GSDME (Abcam), USP47 (Santa Cruz), GAPDH (Abgent Biotechnology), β-actin (Santa Cruz), α-Tubulin (Santa Cruz), Flag (Medical and Biological Laboratories), Myc (Medical and Biological Laboratories), Ub (Santa Cruz), Bax (Santa Cruz).

Whole cell lysates were prepared as described previously [19 (link)]. Equal amounts (30 μg) of total proteins were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, followed by immunoblotting analyses with specific antibodies including antibodies against RNF6 (Thermo Fisher), Flag, Myc, HA (Medical & Biological Laboratories, Tokyo, Japan), ERα, Bcl-xL, Bim-1 (Cell Signaling Technologies., Ltd), or GAPDH (Abgent, Suzhou, China). Anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems.