Catalog no p0013b

The tissues and cells were homogenized using RIPA lysis buffer (Catalog No. P0013B, Beyotime, Shanghai, China) supplemented with protease inhibitors (Catalog No. P1005, Beyotime, Shanghai, China) and phosphoprotease inhibitors (Catalog No. P1081, Beyotime, Shanghai, China). The samples were placed on ice for 1 h with intermittent mixing. The lysates were centrifuged at 13000 rpm for 10 min at 4°C, and the protein content in the supernatants was measured using a BCA protein assay kit (Catalog No. P0012, Beyotime, Shanghai, China). Equal amounts of protein per sample were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST) for 1 h, the membranes were washed thrice with TBST and incubated overnight with primary antibodies at 4°C. The following day, the membranes were washed thrice with TBST for 10 min each time, incubated with the secondary antibody for 1 h at room temperature, and washed thrice with TBST again. The positive bands were developed using an enhanced chemiluminescence detection system (Catalog No. P0018FS, Beyotime, Shanghai, China).

Tissues and cells were lysed in RIPA lysis buffer (Catalog No. P0013B, Beyotime). The lysis was centrifuged, and the supernatant was collected for Western blot analysis. Samples of protein were resolved on SDS‐PAGE and transferred onto PVDF membranes (Catalog No. ISEQ00010, Millipore). The membranes were incubated with the following primary antibodies overnight at 4°C: anti‐Hic‐5 (Catalog No. ab42476, 1:1000, abcam), anti‐α‐SMA (Catalog No. ab5694, 1:500, abcam), anti‐Col1a1 (Catalog No. GB11022, 1:1000, Servicebio), anti‐IL‐6 (Catalog No. GB11117, 1:1000, Servicebio), anti‐NF‐κB/p65 (Catalog No. 10745‐1‐AP, 1:1000, Proteintech), anti‐Vimentin (Catalog No. GB11192, 1:1000, Servicebio) and anti‐GAPDH (Catalog No. 60004‐1‐Ig, 1:5000, Proteintech). Then, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. The following secondary antibodies conjugated to horseradish peroxidase: Affinipure goat antimouse IgG (Catalog No. SA00001‐1, 1:5000, Proteintech) and Affinipure goat anti‐rabbit IgG (Catalog No. SA00001‐2, 1:5000, Proteintech).

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).