Cells of interest seeded on glass bottom petri dishes were fixed with 4% paraformaldehyde for 30 min at 37° and washed three times with 1× PBS. The fixed cells were permeabilized/blocked by incubation with TBST [1× tris-buffered saline and 0.1% Tween 20] containing 5% bovine serum albumin (BSA) overnight at 4°. Primary antibodies were diluted in TBST and 5% BSA solution according to the manufacturers’ recommendations and added to the cells overnight at 4°. The stained cells were washed with 1× PBS and stained with secondary antibody for 1 hour at room temperature.
Polyclonal rabbit and monoclonal mouse anti-Piezo1 was from Novus Biologicals (NBP1-78446 and NBP2-75617); both have been validated by Piezo1 knockdown/knockoff experiments (15 (link), 78 (link)). For all Piezo1 localization quantifications, the monoclonal NBP2-75617 antibody was used. Mouse anti-paxillin antibody was from Merck Millipore (MAB 3060) and rabbit anti-paxillin antibody was from Abcam (ab32115). Mouse anti–activated integrin β1 antibody was from BD Biosciences (9EG7). Rat anti–integrin αvβ3 was from Millipore (LM609).