Comprehensive Western Blot Analysis Protocol

Western blot assays were performed as described previously [25 (link), 26 (link)]. We lysed cells in 1 × SDS loading buffer (Beyotime, China) and incubated at 95–100 °C for 15 min. SDS-PAGE kit (EpiZyme, China) was applied to prepare the gel, and after the electrophoresis in running buffer, we transferred the separated protein lysates to polyvinylidene fluoride membrane. After being blocked in 5% skim milk for 1 h, the membrane then was incubated at 4 °C for 16 h in primary antibodies including anti-Actin (CST, #3700), anti-E-cadherin (CST, #3195), anti-Snail (CST, #3879), anti-Slug (CST, #9585), anti-Akt (CST, #4691), anti-p-mTOR(CST, #5536), anti-CCL2 (R&D Systems, #23007), anti-CD163 (Abcam, #ab87099), anti-p-Akt (CST, #4060), anti-pd-l1 (KleanAB, #P111109) and anti-MAdCAM1 (Proteintech, #21917-1-AP). Next, the membranes were washed and incubated in the secondary antibodies (CST, 1:1000) for 1 h at room temperature. Then the protein signals were visualized by ECL reagents (Millipore, USA) using the ImageQuant LAS 4000 (GE Healthcare) and quantified by ImageJ software.