Ubiquitination Assay for Piezo1 Proteins

Ubiquitination assay protocol is modified from previous work (Zhang et al., 2017 (link); Cao et al., 2021 (link)). WT or mutant Piezo1 transfected cells were lysed in the co-IP buffer (1% w/v CHAPS, 0.6% w/v soy PC, 140 mM NaCl, 1 mM EDTA, 25 mM NaPIPES) supplemented with 2 mM 1,4-Dithiothreitol (Sigma), 1× protease-inhibitor cocktail (Roche) and 20 mM N-ethylmaleimide (E3876; Sigma), and the lysates were centrifuged at 16,000 ×g for 10 min at 4°C; the expression of each protein of interest was confirmed by immunoblotting 5% of the collected supernatants, and the remaining supernatants were incubated with 0.6 μg of anti-GFP antibody and 10 μL of Protein G Dynabeads (ThermoFisher) at 4°C overnight. The recovered beads were washed three times with the co-IP wash buffer (25 mM NaPIPES, 140 mM NaCl, 0.6% w/v CHAPS, 0.14% PC) supplemented with 2 mM 1,4-Dithiothreitol (Sigma), 1× protease-inhibitor cocktail (Roche) and 20 mM N-ethylmaleimide (E3876; Sigma), and heated in 2x sodium dodecyl sulphate (SDS)-PAGE loading buffer containing 1 M urea and 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 62°C for 5 min, and the eluted proteins were immunoblotted with anti-GFP or anti-ubiquitin antibodies.