Fig. 4. Western blot and immunohistochemical localization of Gαs/olf proteins in the trout saccule.

a. Western blot for Gαs/olf protein in the trout saccular hair cell layer. Hair-cell layer lysate was probed with Gαs/olf antibody. Lane 1, molecular size standard (Magic Mark, Invitrogen); Lane 2, trout hair cell layer. The band (arrow) corresponds to the estimated molecular mass for Gαs/olf of 45 kDa, based on the deduced trout Gαs/olf-1/Gαs/olf-2 amino acid sequence. b. Immunohistochemical localizations were conducted with transverse sections cut perpendicular to the longitudinal axis of the trout saccule. At low magnification, Gαs/olf immunoreactivity was localized to the upper two-thirds of the sensory epithelium, with immunostaining of hair cells and neural components. Gαs/olf immunoreactivity was found in hair cell stereocilia throughout the sensory epithelium (arrows) and not observed associated with supporting cells whose nuclei lie just above the basal lamina in the lower third of the epithelium. Scale bar = 50 μm. c-d. At higher magnification, Gαs/olf immunoreactivity was localized to the lateral membrane (intermediate-length arrows) and stereocilia (short arrows) of saccular hair cells. Immunoreactivity was also associated with large afferent fibers (c, single arrowheads encompass large afferent) with circles of immunoreactivity at sites of afferent nerve contacts on hair cells (c, double arrowhead). Small-caliber efferent fibers were immunoreactive for Gαs/olf (d, arrowheads). Scale bar in c, applicable also for d = 10 μm.