Catalog no p0013

Tissues were homogenized with the cell lysis buffer for Western and IP (Catalog no. P0013, Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g for 10 min at 4°C. The resulting supernatants of homogenates (10–40 μg protein/lane) were loaded onto an SDS-PAGE (12.5%), transferred to polyvinylidene difluoride membranes, and incubated with appropriate antibodies (Supplementary Table 2) as previously described (Zhang X. et al., 2020 (link)).

Tissues and cells were lysed in RIPA lysis buffer (Catalog No. P0013B, Beyotime). Nuclear and cytoplasmic protein fractions were extracted from SW1990 cells using a commercial kit (Catalog No. P0027, Beyotime) according to the manufacturer’s instructions. Levels of target proteins were assayed by WB as described^{24 (link)}. The following working concentrations were used for primary antibodies: anti-Flag tag, 1:1000; anti-6*His tag, 1:5000; anti-HA tag, 1:1000; anti-SPOP, 1:500; anti-GFP, 1:1000; anti-β-actin, 1:5000; anti-CDK1, 1:1000; anti-CDK2, 1:1000; anti-E-cadherin, 1:1000; anti-Vimentin, 1:1000; anti-MMP9, 1:1000; anti-ZO-1, 1:1000; anti-ZEB-1, 1:1000; anti-Snail, 1:1000; anti-NANOG, 1:500; and anti-histone H3, 1:10000. Secondary antibodies were used at 1:5000 dilution.
For co-immunoprecipitation assays, cells were lysed in Lysis Buffer for WB and IP (Catalog No. P0013, Beyotime). Lysates were incubated with indicated antibodies at 4 °C overnight on a rocker. Then lysates were immunoprecipitated with Protein A + G Agarose (Catalog No. P2012, Beyotime) for 3 h at 4 °C. Immunoprecipitates were analyzed by western blot, and levels of target proteins were normalized to those of β-actin based on quantitation using ImageJ 1.43 (W. S. Rasband, ImageJ, U. S. National Institutes of Health).