Py925fak

Cells within collagen I gels were washed with ice-cold PBS and then homogenized with truncated pipette tips (3 times for 20 min each on ice) in modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, and 0.25% sodium deoxycholate) with a protease inhibitor cocktail (GenDepot, Barker, TX), as explained in a previous study [26 (link)]. The primary antibodies used were as follows: α-tubulin, α-SMA, talin, and vimentin (Sigma-Aldrich); pY⁴¹⁶c-Src, ERK1/2, phospho-ERK1/2, FLAG, pS⁴⁷³Akt, pS⁴²⁵talin, caspase 3, and Akt (Cell Signaling Technology, Danvers, MA); paxillin, N-cadherin, and FAK (BD Biosciences); pY³⁹⁷FAK, p67LR, laminin, and Twist1 (Abcam, Cambridge, UK); c-Src, pY¹¹⁸paxillin, pY⁵⁷⁷FAK, pY⁸⁶¹FAK, pY⁹²⁵FAK, Snail1, E-cadherin, β-catenin, and Slug (Santa Cruz Biotechnology, Dallas, TX); ZO1 (Zymed Laboratories, Camarillo, CA); vinculin, integrin α6, β1, and β4 (Millipore, Billerica, MA); fibronectin (DAKO, Carpinteria, CA); and KRS (Atlas Antibodies, Stockholm, Sweden).

After lysing cells with extraction buffer, cell extracts were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The following primary antibodies were used: rabbit anti-eNOS, KDR, Cyr61, Myl9, MMP9, ANGPTL3, and VEGF (abcam); EPHB4, RXRα, ERα, pAKT, AKT, pY925 FAK, FAK, pERK, ERK, and mouse anti-GAPDH (Santa Cruz Biotechnology). Antibody incubations were performed overnight at 4 °C. The secondary antibodies used were IRDyeTM-800-conjugated anti-mouse and anti-rabbit IgG (Li-COR Biosciences). Immunoreactivity was detected using an Odyssey Infrared Imaging System (Gene Company Limited). All immunoblots were repeated at least two–three times.