Comprehensive Western Blot Protocol

Western blotting was performed as described previously (34 (link)–36 (link)). Cytoplasmic and nucleic extracts of cells or liver tissues were separated using the protein extraction kit (Beyotime, Shanghai, China) following the manufacturer’s protocols. Intracellular proteins in cells or liver tissues were extracted using cold lysis buffer (25 mM Tris-HCl, PH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS) plus protease inhibitor cocktail and phenylmethanesulfonyl fluoride (PMSF) on ice. The supernatant proteins were concentrated from the cultured media using Amicon Ultra Centrifugal Filter Devices (Millipore, USA) according to the manufacturer’s instructions. The collected protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). Membranes for supernatant proteins were stained with Ponceau S (Sigma-Aldrich) as the loading control. Subsequently, membranes were blocked for 1 h with nonfat dry milk solution (5% in TBS) containing 0.1% Tween-20. The blots were probed subsequently with the indicated primary antibodies and the corresponding secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (Yeasen, Shanghai, China). The antibodies used in this study were listed in Supplementary Table 2.