Immunolocalization of TRPM5 in taste buds of mouse papillae vallatae. The TRPM5-specific antibody Ab-321 recognizes TRPM5 transiently expressed in HEK 293 cells. (A) Western blot analysis of total lysates of mock- (line 1), TRPM5- (line 2) and YFP-TRPM5- (line 3) transfected HEK 293 cells. Positions of TRPM5- and YFP-TRPM5-specific bands are indicated by arrowheads. (B) Overlay of confocal and corresponding differential interference contrast (DIC) images of immunofluorescence staining of HEK 293 cells transiently expressing wild-type TRPM5. (C-E) Immunohistochemistry of taste buds using a TRPM5-specific antibody at lower (C) and higher magnifications (D, E). Notably, TRPM5 is predominantly localized on the basolateral surface of cells (indicated by the arrow in (D, E)). (F) Lack of TRPM5 immunoreactivity in taste receptors after preincubation of the TRPM5-specific antibody with the immunization peptide. (G-I) Fluorescent labeling of taste buds with the TRPM5-specific antibody (E) and the UEA lectin (H) and overlay of both with corresponding DIC image (I). Note that only a subset of cells in the taste bud is enriched in TRPM5. In the TRPM5-positive cells, TRPM5 is mainly localized on the basolateral cell surface and absent in microvilli (as indicated by an arrow (G, H, I)). Scale bars are 20 μm each.