Rabbit anti phospho fak y397

Cells were seeded at 4 × 10⁵ per well in six-well plates with appropriate growth medium and incubated at 37 °C, 5% CO₂ overnight, or treated with drugs for indicated period of time before harvesting. Cell lysates were prepared by cell lysis on ice with 1X RIPA buffer (Cell Signaling, #9806) containing protease inhibitor (Roche, #11836170001) and phosphatase inhibitor cocktail (Sigma, #P5726). Immunoblotting of individual protein bands was performed by incubating the PVDF membranes with the following primary antibodies (all purchased from Cell Signaling) diluted 1:1000 in 5% BSA/TBST: rabbit anti-phospho-ERK (#9101), mouse anti-ERK1/2 (#9107), rabbit anti-phospho-AKT (Ser473) (#4060), mouse anti-AKT (#2920), rabbit anti-phospho-FAK (Y397) (#8556), rabbit anti-FAK (#13009), and rabbit anti-GAPDH (#2118). Fluorescent Alexa 488-conjugated donkey anti-rabbit or anti-mouse antibodies (Life Technologies, A21206 or A21202) were used as secondary antibodies diluted 1:5000 in 5% BSA/TBST to visualize the protein bands on the Pharos Molecular Imager (BioRad). Intensities of individual protein bands from the scanned images were measured using ImageJ after subtracting background. Phosphoprotein levels were then normalized to either GAPDH or total level of the corresponding protein. Uncropped images of immunoblots are shown in Supplementary Figures 7 to 11.

Mouse anti-αv antibody (Abcam, Cambridge, MA, USA, ab16821), rabbit anti-FAK (Cell Signaling Technology, Danvers, MA, USA, #3285), rabbit anti-phospho FAK Y397 (Cell Signaling Technology #3283), rabbit anti GAPDH (Cell Signaling), Anti-mouse Immunoglobulin G-Horseradish peroxidase (IgG-HRP) and anti-rabbit IgG-HRP (Cell Signaling Technology), FAK inhibitors PF-573228 (Sigma-Aldrich, St. Louis, MO, USA) and PF-562271 (Selleckchem, Houston, TX, USA), Osteopontin (R&D System, Minneapolis, MN, USA, #1433-OP-050), 2-NBDG (Thermo Fisher Scientific, Waltham, MA, USA), Lactate Assay kit (BioVision, Milpitas, CA, USA), Oxygen Consumption Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) and MitoTracker Red CMXRos (Invitrogen, suwanee, FA, USA) were purchased. M-PER (mammalian protein extraction buffer), Halt Protease and Phosphatase Inhibitor Cocktail (100×), bicinchroninic acid (BCA) protein assay kit, and culture medium was purchased from Thermo Scientific. Vectashield HardSet Antifade Mounting Solution was from Vector Laboratories (Burlingame, CA, USA).

PEC lysates were prepared with RIPA extraction buffer containing phosphatase inhibitors and protease inhibitors (Roche). Equal amounts of proteins were loaded onto sodium dodecyl sulfate–polyacrylamide electrophoresis gels for separation and transferred onto poly(vinylidene difluoride) membranes. The membranes were blocked with milk and probed with different antibodies: rat anti-CD9 (BD Pharmingen, 553758, 1:1000), rabbit anti-phospho-PDGF receptor-ß Y1009 (Cell Signaling Technology, 4549; 1:1000), rabbit anti-PDGFRβ (Cell Signaling, 3169; 1:1000), rabbit anti-integrin ß1 (Millipore, 04-1109, 1:1000), rabbit anti-CASP3 (Cell Signaling Technology, 9662; 1:1000), rabbit anti-phospho EGFR Y1068 (Cell Signaling Technology, 2234; 1:1000), rabbit anti EGFR (Cell Signaling Technology, 2232; 1:1000), rabbit anti-phospho-FAK Y397 (Cell Signaling 3283, 1:1000), rabbit anti-FAK (Millipore, 06-543, 1:1000). Membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, 7074 and 7076; 1:2000; Jackson Immunoresearch, 706-036-148; 1:1000) and bands were visualized by enhanced chemiluminescence (Clarity Western ECL substrate; Bio-Rad, 170–5061). A LAS-4000 imaging system (Fuji, LAS4000, Burlington, NJ, USA) was used to reveal bands and densitometric analysis was used for quantification. Uncropped blots are shown in Supplementary Figs 18–22.