Protein Lysis and Immunoblotting Analysis

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).

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Hou X., Xia J., Feng Y., Cui L., Yang Y., Yang P, & Xu X. (2021). USP47-Mediated Deubiquitination and Stabilization of TCEA3 Attenuates Pyroptosis and Apoptosis of Colorectal Cancer Cells Induced by Chemotherapeutic Doxorubicin. Frontiers in Pharmacology, 12, 713322.

Publication 2021

RIPA lysis buffer PMSF anti-PARP GSDME USP47 β-actin α-Tubulin Flag Bax

Actin Antibodies Biological Buffer Cultured cells Gapdh Human Protease inhibitor Proteins Ripa Sds page Tissues α tubulin

Corresponding Organization : First Affiliated Hospital of Soochow University

Other organizations : XinHua Hospital, Shanghai Jiao Tong University

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Variable analysis

independent variables

RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 ×protease inhibitor mixture (Roche) and PMSF (Beyotime, Shanghai, China)

dependent variables

Protein expression levels of PARP, GSDME, USP47, GAPDH, β-actin, α-Tubulin, Flag, Myc, Ub, and Bax

control variables

Not explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

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