Twist ab50887

Cells were lysed on ice in RIPA (radio-immunoprecipitation assay) buffer with 1 mM PMSF (phenylmethylsulfonyl fluoride). From the cell lysate, 40 μg of total protein was loaded into the wells of the SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel elec trophoresis) gel, along with a molecular weight marker. Electrophoresis was carried out for 1–2 h at 100 V, followed by transfer to PVDF (polyvinyl difluoride) membranes, which were then blocked with 5% BSA (bovine serum albumin). The membranes were then incubated overnight at 4°C with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, vimentin sc-6260, Santa Cruz; fibulin-4 ab125073, Snail ab167609, Slug ab27568, Twist ab50887, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT 38449, mTOR ab32028, p-mTOR ab109268; Abcam) at working dilutions of 1:1000. After washing the membranes thrice with TBST for 5 min each, the membranes were incubated with conjugated secondary antibody diluted to 1:1000, at room temperature for 1 h. Blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).

Using radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride, cells were lysed on ice. Protein samples (40 μg/lane) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinyl difluoride membranes and blocked with 5% bovine serum albumin. These membranes were then cultured with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, vimentin sc-6260, Santa Cruz; HMGB1 ab18256, Snail ab167609, Twist ab50887, Abcam) with working dilutions 1:1,000 at 4°C overnight. The next day, these membranes were incubated with secondary antibody with working dilutions 1:2,000 at room temperature for 1 h, and blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).