Ma5 32876

Cells were washed with PBS and lysed directly in the cell culture dish with 80 μl of 1x reduced Laemmli buffer (Fisher Scientific 15493939) and boiled for 5 min at 95°C. 15 μl of each sample was loaded onto a 4-15% Mini-PROTEAN TGX stain-free protein gel (Bio-Rad 4568086). Total protein was analysed using the Criterion Stain-free imaging system (Bio-Rad) and subsequently transferred on polyvinylidene difluoride membranes using the Trans-Blot-Turbo Transfer System (Bio-Rad). Membrane blocking was carried out with 5% nonfat dry milk/TBS-T for 1 h at room temperature. The primary antibodies targeting PIEZO1 (Thermo Fisher Scientific MA5-32876, 1:500) and β-tubulin (MERCK Millipore MAB3408, 1:10’000) were diluted in 5% BSA/TBS-T and incubated overnight at 4° C. Next, the membranes were washed 3 times in TBS-T and incubated with the secondary antibody (anti-mouse, Sigma-Aldrich SAB3701073, 1:20’000) for 1 h at room temperature. Images were taken using UltraScence Pico Ultra Western Substrate (GeneDireX CCH345-B) and the ChemiDoc MP imaging system (Bio-Rad).