Cellular localization of wild-type and mutant PIEZO1 determined by IF confocal microscopy. (A) Wild-type and mutant FLAG-tagged PIEZO1-expressing HEK293 cells were transfected with ER-GFP, a plasmid that expresses GFP fused to the ER signal sequence of calreticulin and KDEL (ER retention signal). Cells were stained with anti-FLAG AF555 (red) and anti-GFP (green). (B) Wild-type and mutant FLAG-tagged PIEZO1-expressing HEK293 cells were stained with anti-FLAG ATT0647N (red), DAPI (blue), and an antibody against Golgi apparatus, anti-GM130, conjugated with Alexa Fluor 488 (green). Three arginine mutants, R2488Q, R2302H, and R2088G, demonstrated partial intracellular retention, colocalizing with ER and Golgi markers. Scale bars, 10 μm.