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Fig. 1
UPRT expression and activity in zebrafish hair cells. a Diagram of the construct used for Tol2 Gateway-mediated transgenesis. A minimal myo6b promoter was used to drive expression of the HA-UPRT-P2A-mCherry transgene in auditory, vestibular, and lateral line hair cells. b-b'' Maximum projection images of immunolabeled HA-UPRT and fixed mCherry fluoresecence in inner ear hair cells of a 3 dpf Tg(myo6b:UPRT) larva. AC, anterior crista; AM, anterior macula; LC, lateral crista; PC, posterior crista. The focal plane includes a neuromast (NM-MI1). c Dot blot for TU-tagged, biotinylated total RNA demonstrating the enzymatic activity of UPRT in the hair cells of 5 dpf zebrafish larvae. Nontransgenic wild-type (WT) larvae exhibited low levels of 4TU incorporation in contrast to Tg(myo6b:UPRT) larvae when exposed to 5 mM 4TU for 3 h. RNA from Tg(myo6b:UPRT) larvae exposed to DMSO only did not exhibit any detectable biotinylation