Mab5416

The brain tissues were homogenized in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany) on ice and stored at −80°C until use. Protein concentrations in the supernatant were determined using a BCA assay kit (Beyotime, Shanghai, China). The proteins were separated through the polyacrylamide SDS gels and transferred to 0.45 μm PVDF membrane (Millipore, Darmstadt, Germany). After blocking with 5% non-fat milk, the membranes were incubated with a primary antibody at 4°C overnight. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (anti-mouse: 1:5,000, 7076S, Cell Signaling; anti-rabbit: 1:5,000, 7074S, Cell Signaling) after washing with TBST (TBS containing 0.2% Tween-20). Then, immunoreactivity was detected by a chemiluminescent reagent (Amersham-GE, Pittsburgh, United States). The following primary antibodies were used (dilution, source): TRPV1 (1:1,000, NB100-1617, Novus Biologicals, Littleton, CO), GAPDH (1:3,000, 2118S, Cell Signaling), GluA1 (1:1,000, AB1504, Millipore), GluA2 (1:1,000, 13607S, Cell Signaling), GluA3 (1:500, MAB5416, Millipore), Src (1:1,000, 2110S, Cell Signaling), p-Src (Tyr416) (1:1,000, 2101S, Cell Signaling), p-Src (Tyr527) (1:1,000, 2105S, Cell Signaling), cofilin (1:1,000, 5175S, Cell Signaling), and p-cofilin (1:1,000, 3313S, Cell Signaling).