Functional characterization of DrAqp3b mutants. (A) Pf of control oocytes (water-injected) and oocytes expressing 1 ng cRNA encoding wild-type DrAqp3b (DrAqp3b-WT) or different DrAqp3b mutants at different pH. Values are the mean ± SEM of three experiments (n = 8-10 oocytes per construct). The asterisks denote significant differences between WT and mutant DrAqp3b at a given pH (Student′s t test, p < 0.05). (B) Immunofluorescence microscopy of water-injected oocytes (control), or oocytes expressing DrAqp3b-WT, -H53A, -H53A/G54H, -T85A and -H53A/G54H/T85A using an affinity purified anti-DrAqp3b antiserum. The arrowhead points to the oocyte plasma membrane. (C) Immunoblot of total membrane protein extracts of control oocytes or oocytes expressing DrAqp3b-WT treated or not with N-glycosidase F (PNGase F). The arrows indicate glycosylated and deglycosylated forms of DrAqp3b-WT. (D) Representative immunoblot of plasma membrane protein extracts of oocytes expressing increasing amounts of cRNA (1-8 ng) encoding DrAqp3b-WT, -T85A and -H53A/G54H/T85A treated with PNGaseF.