Mouse anti α actinin antibody

The following commercially available antibodies were used: anti-GFP antibody (Santa Cruz Biotechnology, Dallas, TX, United States; 1:5,000 dilution); mouse monoclonal anti-Piezo1 antibody (Cat# NBP2-88938, Novus Biologicals, Centennial, CO, United States; 1:1,000 dilution); mouse anti-α-actinin antibody (Santa Cruz Biotechnology; 1:5,000 dilution); mouse anti-Na⁺/K⁺ ATPase antibody (a6F, DSHB; 1:1,000 dilution); mouse anti-ubiquitin antibody (clone FK2, ENZO, 1:1,000); rabbit anti-GAPDH antibody (CellSignaling, 1:1,000); anti-rabbit IRDye680 (Li-Cor, 1:20,000); anti-mouse IRDye800 (Li-Cor, 1:20,000); mouse anti-HA antibody (Sigma, 1: 200 for IF); Alexa Fluor 555 anti-mouse (ThermoFisher, 1:200 for IF). Unless specified, all the antibody dilutions refer to western blotting.

Transiently transfected cells were solubilized in a modified RIPA buffer [Tris buffer 10 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, NaCl 140 mM, in (% w/v): Sodium deoxycholate 0.1, SDS 0.1, Triton X-100 1.0, pH 7.2] supplemented with 1× EDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich), 1 mM (phenylmethylsulfonyl fluoride) PMSF, 10 mM TCEP, and 1 mM N-ethylmaleimide (NEM) for 10 min on a rotating wheel at 4°C. Cell lysates were cleared by centrifugation at 13,000 ×g at 4°C for 10 min. For quantitative western blot analysis, GFP-fused Piezo1 was probed with a rabbit monoclonal anti-GFP antibody (Santa Cruz Biotechnology, Dallas, TX, United States); or a mouse monoclonal anti-Piezo1 antibody (Cat# NBP2-88938, Novus Biologicals, Centennial, CO, United States); mouse anti-α-actinin antibody (Santa Cruz Biotechnology) was added simultaneously for a loading comparison followed by anti-rabbit IRDye680 and anti-mouse IRDye800 (Li-Cor) to enable quantification with the LI-COR Odyssey system (LI-COR Biotechnology, Lincoln, NE, United States).