Anti actin a5316

All chemicals were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). The antibody for p16 (Sc-1661), p21 (Sc-6246), p27 (Sc-1641), MMP-2 (sc-10736), NFκB (Sc-109), IκB (Sc-847), ubiquitin (Sc-8017), Ras (Sc-32), RhoA (Sc-418), RhoB (Sc-8048), CDC42 (Sc-87), VEGF (Sc-4570), and AKT (Sc-5298) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). The antibodies for FAK (CST#3285), PI-3K (CST#4255), phosphorylated AKT (CST#9275,), phosphorylated FAk (CST#3281), and integrin β3 (CST#4702) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-actin (A-5316,) was purchased from Sigma-Aldrich Company.

Cell pellets were lysed with SDS Lysis Buffer (1% SDS, 10 mM Tris pH 7.4, 1 mM phenylmethylsulfonyl fluoride) and supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Protein samples were resolved by SDS-PAGE (Novex NuPage Bis Tris Gel; Invitrogen MiniGel Tank) and then transferred onto PVDF membranes (iBlot 2 system, Invitrogen). The following antibodies were used: KLF4 D1F2 from Cell Signaling, anti-Actin A5316 from Sigma, Direct-Blot™ HRP anti-β-actin Antibody, GAPDH Loading Control Monoclonal Antibody (GA1R), PARP antibody from Cell Signaling, and MYC antibody from Cell Signaling. Primary antibodies were used at 1:1,000 dilution. Primary actin antibody was used at 1:100,000 dilution, or HRP anti-β-actin at 1:300,000. HRP cross-linked secondary antibodies (anti-rabbit IgG, HRP-linked Antibody #7074 and anti-mouse IgG, HRP-linked Antibody #7076 from Cell Signaling) were detected by West Femto Maximum Sensitivity Substrate (Thermo Fisher) and Amersham Hyperfilm ECL (GE). All secondary antibodies are used at 1:15,000-1:30,000 dilution. Protein quantity was normalized based on housekeeping control bands.