Western Blot Analysis of Sodium Channel Proteins

30μg of proteins were separated by SDS-PAGE on 10% acrylamide gels, and proteins were electrically transferred to PolyScreen PVDF hybridization transfer membranes (Perkin Elmer, Boston, MA). Membranes were incubated overnight at 4°C with primary rabbit antibody for Scnn1a (1:500), Scnn1b and Scnn1g (1:1000) [32 (link)], CAP2/Tmprss4 [33 (link)] (1:200) and β-actin (1:1000, Sigma-Aldrich) and for 1 hour with donkey anti-rabbit IgG HRP-conjugated secondary antibody (1:10000, Amersham, Burkinghampshire, UK) (all antibodies in TBS-Tween 1% and dried milk 2%). The signal was revealed using SuperSignal West Dura detection system (Pierce, Rockford, IL) and quantified using ImageStudio^TM Lite program (LI-COR). Kidney extracts from inducible renal tubule-specific Scnn1a KO mice, generated by interbreeding of Scnn1a^lox/lox mice [34 (link)] and Pax8::rtTA/LC1 mice [35 (link)], were used as control for Scnn1a-specific signals on Western blot (control non-doxycycline-induced animals [Ctrl WT], control doxycycline-induced animals [Ctrl KO]). The same strategy was applied for Scnn1b- and Scnn1g-specific bands [36 ]. The specificity of the primary antibody for CAP2/Tmprss4 has been described previously and extensively tested in vitro using the Xenopus oocyte expression system [33 (link)], and corroborated using protein extracts from CAP2/Tmprss4 knock-out mice that were used as control.