3T3 cells were fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.1% Triton X-100 for 10 min. The cells were then blocked with 5% goat serum and incubated with anti–phospho-FAKY397 antibody (Abcam, UK) and vinculin antibody (Cell Signaling Technology, USA) at 4°C overnight. Then, a DyLight 488 goat anti-rabbit polyclonal antibody (Zuangzhibio, China) was used as secondary antibody. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. FAKY397 phosphorylation and vinculin were detected after 3 days of cell culture. ImageJ software was used to quantify cell adhesions from imaging data. Briefly, the pixel intensity was extracted for each adhesion, which was then converted to a length in ImageJ by selecting the cell adhesion profile for each adhesion and setting minimal adhesion size to 0.15 μm2. Last, cell adhesion areas were calculated using ImageJ.

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