Mutation of I696X and W697X does not abrogate receptor surface expression. (A and B) Total and surface expression of representative I696X (A) and W697X (B) mutants. TRPV1 species were transiently expressed in HEK293 cells. At 48 h, cells were harvested, membrane proteins were biotinylated and purified by agarose-Streptavidin, and protein expression was analyzed by Western immunoblotting using a polyclonal anti-TRPV1 antibody. Whole cell and avidin-purified extracts were separated in 10% SDS-PAGE gels, electrotransferred to a PVDF membrane, and probed with the anti-TRPV1 antibody. Immunoblots were visualized with the ECL system. C and D denote the quantification expressed as the surface/total protein ratio of bands shown in A and B. Actin was used as a loading control. Mutants were chosen to represent the amino acid families. Data represent mean ± SEM (error bars), with n (number of independent experiments) = 3. *, P < 0.05 as compared with wild type, using a Student’s t test.