Fibulin 3 sc 33722

Target cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor (RIPA:PMSF = 100:1). After determining the protein concentration by the Bicinchoninic Acid (BCA) method, protein samples (40 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 h with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin (BSA). The membranes were then incubated on a shaking bed with primary antibodies (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab106077, Twist ab50887, Zeb 2 ab138222, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT ab38449, mTOR ab32028, p-mTOR ab109268, Abcam; p-ERK (Thr202/Tyr204) #4370, ERK #9102, Cell Signaling), at a 1:2,000 dilution overnight at 4 °C. The next day, the membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Finally, positive labeling of the proteins on the membranes was visualized by enhanced chemiluminescence (ECL) using a Bio-Rad ECL kit (Solarbio).

Cells were lysed on ice in RIPA (radioimmunoprecipitation 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 electrophoresis) 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 (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab27568, Twist ab50887, β-catenin ab32572, C-myc ab32072, Cyclin D1 ab134175, 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.).