Anti vinculin

ERK1/2 phosphorylation experiments were carried out on transfected HEK293T cells that had been seeded in 6-well plates (70 percent to 80 percent confluence). Following transfection, cells were incubated for 48 h under serum-starved conditions and treated with 20 ng/mL epidermal growth factor (EGF) for 5 or 15 min or left unstimulated. Using a radioimmunoprecipitation technique, the total protein was recovered from the lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Transfer membranes containing equal amounts of proteins were treated overnight at 4°C with specified primary antibodies and then at 37°C for two hours with secondary antibodies. The primary antibodies and dilutions were as follows: anti-SHP2 (1 : 3000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-pERK1/2 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), and anti-Vinculin (1 : 5000, Abmart, Shanghai, China). Anti-rabbit and anti-mouse horseradish peroxidase- (HRP-) conjugated secondary antibodies were employed at 1 : 5000. Band density was quantified with ImageJ software.

The cells were lysed in RIPA lysis buffer (Servicebio, China) containing 1% protease inhibitor for 30 minutes, and the protein supernatant was obtained after centrifugation. The supernatant was then mixed with 5× protein loading buffer (Coolaber, China) and heated at 100°C for 10 minutes in preparation for sample loading. Electrophoresis was carried out by SDS-PAGE and the proteins were then moved to NC membrane. The membranes were blocked with TBST buffer containing 5% skimmed milk powder for 2 hours and then placed in a 4°C shaking table overnight with various primary antibodies. The next day, the membrane was incubated with secondary antibody at room temperature for one hour. A Clinx Gel Documentation and Analysis instrument was used to observe the protein bands, and ImageJ was used to analyze the gray value of the bands. The antibodies used were as follows: anti-cyclin D1 (Cell Signaling Technology, USA, 1:1000), anti-CDK4 (Cell Signaling Technology, USA, 1:1000), anti-CDK6 (Cell Signaling Technology, USA, 1:1000), anti-vinculin (Abmart, China, 1:1000), anti-β-tubulin (Cell Signaling Technology, USA, 1:1000), goat anti-rabbit secondary antibody (Cell Signaling Technology, USA, 1:5000), and goat anti-mouse secondary antibody (Cell Signaling Technology, USA, 1:5000).