Integrin αv

Cell lysate was prepared using lysis buffer consisting of 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.5% deoxycholate, 1% NP-40, 0.3% SDS, and 1 mM phenylmethylsulfonyl fluoride.²⁹ (link) Immunoblot analyses were performed according to the procedure described previously.²⁹ (link) The antibodies used were specific for human STAT3, phosphorylated STAT3 (EMD Millipore, Billerica, MA, USA), HSP90α (AbD Serotec), phosphorylated FAK (Invitrogen), phosphorylated CDK2 (Epitomics, Burlingame, CA, USA), phosphorylated Rb, Rb, phosphorylated CDC2 (Cell Signaling, Danvers, MA, USA), connexin-43 (Sigma, St. Louis, MO, USA), integrin α_V (BD Biosciences, San Jose, CA, USA), cyclin D1, MMP-2, MMP-9 (Lab Vision/NeoMarkers Co., Fremont, CA, USA), CDK4, cyclin E, cyclin A, CDK2, cyclin B1, CDC2, FAK, TCF12, Twist-1, Snail, Slug, E-cadherin, connexin-26, fibronectin, and GAPDH (Santa Cruz Biotechnology).

Whole-cell lysates were extracted using radioimmunoprecipitation assay buffer, and protein concentration was determined using the BCA protein assay kit (Pierce, Grand Island, NY, USA). Equal levels of protein from the samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), and the separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and blocked with Tris-buffered saline-Tween-20 with 5% dry skimmed milk for 1 h at room temperature. To assess protein expression, the blots were incubated with the following primary antibodies at 4 °C overnight: rabbit antibodies against His-tag, E-cadherin, ZO-1, β-catenin, N-cadherin, VIM, Snail, and Slug (Cell Signaling Technology, Danvers, MA, USA); and mouse antibodies against Flag (Sigma-Aldrich, St Louis, MO, USA), BPIFB1 (Abnova, Taipei, Taiwan); VTN and integrin αV (BD Biosciences). After washing, the blots were incubated with horseradish peroxidase-labelled secondary antibodies (Cell Signaling Technology) and detected by enhanced chemiluminescence (EMD-Millipore). GAPDH (Cell Signaling Technology) served as an endogenous control for equal loading.