HeLa cells were transfected with pEGFP-N3-tagged constructs and GFP-positive cells were sorted by FACSAria Sorter (BD) after 24 hr to obtain the cells with a comparable expression level of GFP-constructs, and then were transferred to fibronectin (12.5 μg/ml)-coated coverslips. After 2 hr, the cells were fixed with 4% paraformaldehyde (PFA, for 15 min at 37°C). After washing with PBS, the cells were treated with 0.1 Triton X-100 for 10 min at room temperature and blocked in 2% bovine serum albumin. The FAs were stained by anti-Paxillin antibody (Mouse, BD Bioscience), followed by Alexa Fluor 594 donkey anti-mouse secondary antibody (Thermo Fisher Scientific) and the F-actin was stained by Alexa Fluor 647 phalloidin (Thermo Fisher Scientific). The cells were visualized with 100× objective using a Nikon A1R HD25 Confocal Microscope.

In cell spreading assays, the cells were stained by Alexa Fluor 594 phalloidin (Thermo Fisher Scientific) for the quantification of cell area, and the cells were visualized with 20× objective using a Leica DMI6000B microscope. The cell areas of GFP-signaling positive cells were analyzed.

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