Anti asic3 antibody

Frozen specimens from rodents were homogenized in 100 μl lysis buffer containing a mixture of proteinase and phosphatase inhibitors and then centrifuged at 15 000 rpm for 15 min at 4°C. The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). A total of 50 μg of protein was resolved on 12% precasted SDS‐PAGE gels, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% non‐fat milk in PBS containing 0.1% Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies. The following antibodies were used in this study: GAPDH, anti TNF‐α, anti‐ASIC1, and anti‐ASIC3 antibody (1:1000, abcam, USA). After washing with Tris‐Buffered Saline, 0.1% Tween 20 Detergent (TBST), the blots were incubated for 2 h at room temperature with HRP‐conjugated secondary antibody (1:5000; Amersham Biosciences, San Francisco, CA, USA), visualized by using Electro‐Chemi‐Luminescence (ECL) chemiluminescent detection system (Amersham Biosciences).