Immunoblotting of TRPC Proteins

These methods were described previously [13 (link)]. Renal cortex was homogenized in M-PER™ mammalian protein extraction buffer (Thermo Fisher Scientific) and sonicated on ice. The homogenates were subjected to centrifugation at 13,000 rpm for 30 min. Protein concentrations of the supernatants were determined with Bradford reagent (Bio-Rad). Rabbit antibodies against TRPC6 (ACC-017) and TRPC3 (ACC-016) were from Alomone Labs. The TRPC6 antibody targets motifs encoded by exon 1 located in the amino-terminal and upstream of the deletion in the Trpc6^del allele. We also used a polyclonal TRPC6 antibody from Booster Biological Technology that targets residues 248–264 in the rat channel, downstream of the deletion. A mouse monoclonal antibody against TRPC5 was from NeuroMAB. We also used a rabbit polyclonal antibody against TRPC5 from Alomone (ACC-020). All TRPC antibodies were used at a dilution of 1:1000. A mouse monoclonal antibody (clone 1A4) against α-smooth muscle actin (SMA) was from Sigma-Aldrich.