Anti fak

Following 72 h of treatment, total protein was extracted from CAL 27 cells cultured on poly-HEMA using RIPA Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease and phosphatase inhibitors (Roche, Mannheim, Germany). Equal amounts of protein samples were separated through SDS-PAGE and transferred onto nitrocellulose membrane BioTrace™ NT (Pall Life Sciences, Ann Arbor, MI, USA), followed by blocking. The primary antibodies were used to probe the blots at 4 °C overnight: anti-αv integrin (1:5000; Cat#ab179475, Abcam), anti-phosphorylated FAK (Tyr397, D20B1, 1:1000; Cat#8556, Cell Signaling Technology Inc., Santa Cruz, CA, USA), anti-FAK (1:1000; Cat#3285, Cell Signaling Technology Inc.), anti-phosphorylated Src (Tyr416, D49G4, 1:1000; Cat#6943, Cell Signaling Technology Inc.), anti-Src (36D10, 1:1000; Cat#2109, Cell Signaling Technology Inc.), and anti-β-actin (1:1000; Cat#TA-09, Zhongshan Goldenbridge Biotechnology). Next day, the membranes were washed and probed with fluorophore-conjugated anti-mouse (1:10,000; Cat#P/N 925-32210, LI-COR Biosciences, Lincoln, NE, USA) and anti-rabbit (1:10,000; Cat#P/N 925-32211, LI-COR Biosciences) secondary antibodies at room temperature for 1 h. Blots were scanned with Odyssey® Imaging system (LI-COR Biosciences) and analyzed using LI-COR Image Studio Software 4.0 (LI-COR Biosciences) with β-actin as an internal control.