Sds page kit

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.

Cellular protein of osteoblasts from rats was extracted with RIPA lysis buffer (#P0013B, Beyotime, China), and BCA kit (#P0012S, Beyotime, China) was adopted to determine the protein concentration. Subsequently, the SDS-PAGE gels were prepared according to the SDS-PAGE kit (#PG112, Epizyme, China). The electrophoresis condition was set at 80 V for 1 h and then was adjusted to 120 V for 40 min. The membrane transfer condition was settled at 250 mA for 2 h. Then, 5% skimmed milk was used for blocking at 25°C for 1 h. Primary antibodies PTGS2 (#A1253, ABclonal, China), β-catenin (#A19657, ABclonal, China), TCF4 (#A1141, ABclonal, China), ALP (#A19286, ABclonal, China), and GAPDH (#A19056, ABclonal, China) (dilutions of 1 : 1000, 1 : 500, 1 : 500, 1 : 500, and 1 : 100, respectively) were added for incubation overnight at 4°C. The membranes were washed 3 times with TBST, 10 min each time. Next, secondary antibody (#AS014, ABclonal, China) was added to the membranes and incubated at room temperature for 1 h. Then, the membranes were rinsed with TBST 3 times, 10 min each time, and exposed using ECL exposure liquid. The gray values of the target bands were analyzed and calculated by ImageJ (version 1.8.0, USA).