Expression of PEZO-1 in Sf9 Cells

To express PEZO-1 in Sf9 cells (a clonal isolate of Spodoptera frugiperda Sf21 cells), we generated recombinant baculoviruses, according to the manufacturer's instructions (Bac-to-Bac expression system; Invitrogen). To generate this baculovirus, we used a pFastBac construct (Epoch Life Science) containing an 8× histidine–maltose binding protein tag and a synthesized pezo-1 isoform G nucleotide sequence (one of the longest isoforms according to RNA sequencing; wormbase.org release WS280). For expression of PEZO-1 R2405P, the construct contained an 8× histidine–maltose binding protein tag and a synthesized pezo-1 isoform G with the R2405P point mutation. We infected Sf9 cells with either wild-type or mutant pezo-1 baculovirus for 48 h as previously described (Millet et al., 2022 (link)). Infected cells were plated on glass coverslips coated with a peanut lectin solution (1 mg/ml; Sigma-Aldrich) for patch-clamp experiments.