FIGURE 2.

Co-IP of TRPA1 and TRPV1 from trigeminal neurons. A, immunolabeling of rat TG frozen sections (20 μm thick) with rabbit polyclonal anti-TRPA1 N-terminal antibody (marked TRPA1) and a mix of anti-TRPA1 and 100 μg/ml blocking peptide (marked TRPA1+BP). Anti-TRPA1 recognizes an N-terminal epitope with the amino acid sequence KRSLRRVLRPEERKE, which serves as a blocking peptide (BP). B, TG lysate (50 μg) was immunoprecipitated with anti-TRPA1 and labeled (WB) with anti-TRPA1 antibody mixed with vehicle (bottom) or blocking peptide (top). Anti-TRPA1 antibody was preincubated with 100 μg of BP. C, control for TRPA1 co-IP. TG lysate was immunoprecipitated with TRPV1 (V1), non-immune serum (Ser), and protein A-agarose beads (Bead) and then labeled with anti-TRPA1 antibodies. D, co-IP of TRPA1 and TRPV1 from cell extract of rat WT or TRPV1 KO TG neurons. Samples not processed by co-IP cell lysates (marked with a minus sign) were derived from TG neurons of corresponding animal lines and were used as loading and positive controls. Antibodies for IP are indicated below the lanes. A1, antibodies against TRPA1; V1, antibodies against TRPV1. Western blot (WB) antibodies are indicated at the right. Molecular mass markers are shown.