Anti α actinin antibody

HEK293 cells were transiently transfected in 24-well plates using Liposome-based transfection reagent Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) to deliver PIEZO1 cDNA into the cells. Cells were lysed in a lysis buffer containing 1% NP-40 (Sigma-Aldrich, Castle Hill, NSW, Australia) and protease inhibitors (Roche, Cromer, NSW, Australia) after 48 h and equal volume of lysate loaded for SDS–PAGE and western blot analysis. The nitrocellulose membranes were probed simultaneously with a rabbit polyclonal anti-GFP antibody at a 1:5,000 dilution (Abcam, Cambridge, UK; Cat no—ab290) and a mouse monoclonal anti α-actinin antibody at 1:1,000 dilution (Santa Cruz Biotechnology, Dallas, TX, USA Cat no—sc17829) overnight. Both anti-mouse IRDye800 and anti-rabbit IRDye680 (Li-Cor Biotechnology, Lincoln, NE, USA) at a 1:20,000 dilution were incubated with the membrane and the PIEZO1 proteins were detected using the Li-Cor Odyssey system (Li-Cor Biotechnology). Western blot images were produced using ImageStudioLite (Li-Cor Biotechnology).

The cells were plated on culture slides and fixed in 4% paraformaldehyde for 30 min, washed with PBS, and stained with an anti-α-actinin antibody (Santa Cruz) in immunofluorescence buffer at 4 °C overnight. Then, the sections were washed with PBS and incubated with the secondary antibody (Abways) for 90 min. The nuclei were stained with Hoechst. Images were obtained using a fluorescence inverted microscope (Olympus) and analyzed with ImageJ.