FAKY397 phosphorylation was detected after 2 hours of cell seeding. Total proteins were extracted with a mammalian protein extraction kit (CWBIO, China) according to its protocol. The protein samples were separated by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% bovine serum albumin at room temperature for 1 hour, followed by incubating with anti–phospho-FAKY397 and anti-FAK antibody (1:1000; Abcam, UK) at 4°C overnight. After washing with triethanolamine buffered saline and polysorbate 20 (TBST), the membranes were incubated with secondary antibodies (1:5000; Cell Signaling, USA) at room temperature for 1 hours. The relative integrated density of each protein band was determined using an Odyssey infrared imaging system (LI-COR, USA).

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