Biotinylation assay and Western blotting

Biotinylation of plasma membrane proteins was performed using a slightly modified protocol from a commercially available cell surface protein isolation kit (BioVision). CHO-K1 cells were first transfected with ASIC3-TUR or ASIC1a-CER with and without unlabeled STOM. Approximately 18 h after transfection, cells were quick-washed in ice-cold PBS followed by incubation with Sulfo-NHS-SS-Biotin at 4°C with gentle shaking. After 1 h of biotin labeling, reaction was quenched, and cells were scraped and collected followed by centrifugation at 1,000 ×g for 5 min. Cells were washed twice in Tris-buffered saline (TBS) buffer followed by centrifugation at 1,000 ×g for 5 min. Pellet was collected, and cells were resuspended in RIPA buffer for 1 hr at 4°C with end-over-end mixing (in mM): 150 NaCl, 50 Tris, 1% NP-40, 0.5% sodium deoxycholate, and 1X protease inhibitor added before lysis (Thermo Fisher Scientific, Pierce). Lysed cells were centrifuged at 10,000 ×g for 7 min, and supernatant was transferred onto packed streptavidin beads and incubated with end-over-end mixing for 1 h at room temperature. A portion of each sample was also collected before loading onto beads to quantify total protein concentration. Beads were centrifuged at 800 ×g for 60 s, and supernatant was collected as the non–biotin-bound (intracellular) fraction. Beads were washed three times in TBS followed by centrifugation at 800 ×g for 60 s. Biotin-labeled protein bound to streptavidin beads was eluted by incubating beads with 100 mM DTT for 1 h at room temperature. Sample was centrifuged at 800 ×g for 60 s, and supernatant was collected as biotinylated (plasma membrane) protein.

Samples that were collected just after lysis were measured for the total protein concentration using a Bicinchoninic Acid (BCA) assay kit (Thermo Fisher Scientific, Pierce). Both the biotinylated and intracellular fractions were normalized to total protein and loaded on a 4–12% Bis-Tris precast gel (Thermo Fisher Scientific, Invitrogen) and run at 200 V for 30 min. Protein was transferred from gel to a PVDF membrane at 100 V for 1 hr. Membrane was incubated in blocking buffer for 1 h followed by overnight incubation in primary antibody (purified rabbit anti-GFP; Torrey Pines Biolabs) at 4°C. Membrane was then washed six times with TBS-T (Tris-buffered saline + Tween20) followed by 1-h incubation with secondary antibody (goat anti-rabbit IgG; KPL). Membrane was washed another six times in TBS-T and then developed in the dark for 5 min with chemiluminescent reagent (Immobilon Forte; Millipore).