6), 1% Triton X-100, and 1 Complete protease inhibitor tablet (Roche Applied Science) per 50 ml] in a selleckchem 1.5-ml tube with an Eppendorf micropestle. Homogenized samples were centrifuged for 10 min at 12,000 rpm, and the middle liquid layer was retained for gel electrophoresis. Protein concentrations were determined with a BCA assay kit (Pierce, Rockford, IL). Electrophoresis and immunoblot analysis. Equivalent amounts of protein (30 ��g) from the oocyte total membrane preparations were boiled in 1�� Laemmli sample buffer [62.5 mM Tris?HCl (pH 6.8), 25% glycerol, 2% SDS, 5% ��-mercaptoethanol, and 0.01% bromophenol blue] at 95��C for 5 min and subjected to SDS-PAGE with an 8% separating gel and a 4% stacking gel. Proteins were electrotransferred to methanol-treated polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) for 1.
5 h at 100 V at 4��C in a Tris-glycine buffer [25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol]. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) buffer [100 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween] for 1 h at room temperature. Membranes were incubated overnight at 4��C with rabbit polyclonal antibody to ASIC1 (Alomone Labs, Jerusalem, Israel) at 1:200 or with mouse anti-actin (Chemicon, Billerica, MA) at 1:5,000 in 2% milk in TBST. Primary antibody incubation was followed by three washes of 15 min each with TBST. A goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (Pierce) diluted 1:10,000 or a goat anti-mouse IgG HRP-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:20,000 in 2% milk in TBST was used to detect the primary antibodies for 1 h at room temperature.
After three 15-min washes with TBST, membranes were treated with SuperSignal West Pico Substrate (Pierce) and exposed to Kodak Biomax XAR film (Perkin-Elmer, Waltham, MA). Single oocyte chemiluminescence. To quantify the expression of hASIC1b at the plasma membrane of oocytes in control or PKC-modulated conditions, we used the method of single oocyte chemiluminescence (SOC) as described by Zerangue et al. (45). Oocytes expressing HA-tagged WT hASIC1b were treated with PMA (1 ��M for 5 min), PdBu (1 ��M for 5 min), chelerythrine (1 ��M for 1 h) or both chelerythrine (1 ��M for 1 h) and PMA (1 ��M for 5 min), as was done for recording peak pH 4.0-activated currents.
They were then fixed in 4% paraformaldehyde in ND96 pH 7.4 solution for 15 min at 4��C. All subsequent steps were performed at 4��C. Oocytes were washed three times for 5 min each time in ND96 pH 7.4 solution and incubated in blocking solution (1% BSA in ND96 pH 7.4 solution) overnight. They were then incubated in rat anti-HA antibody (clone 3F10, Roche) at 1 ��g/ml for 2 h. Carfilzomib They were washed twice in blocking solution for 30 min each time and incubated in goat anti-rat HRP-conjugated secondary antibody (Pierce) at 1:400 for 1 h.