Controls of zebrafish and yeast experiments. A-B: RT-PCR with cDNA of 6 different developmental stages (from 1,25 to 72 hpf) amplifies a 1000 bp band (arrow) as expected. vps15 mRNA is maternally expressed before midblastula transition (1,25 hpf) and mRNA is detectable until at least 72 hpf. PCR without prior reverse transcription did not yield a 1000 bp band (B) indicating that the amplified fragment is not due to contamination with cDNA or genomic DNA. C: In situ hybridization with vps15 antisense probe in zebrafish embryos showing ubiquitous expression at 14 hpf and prominent localization in the head as well as in the pronephric duct (pd) at 48 hpf. Ov: otic vesicle. D. Co-injection of vps15-mo with zVps15-GFP plasmid completely blocked GFP expression. As expected, co-injection of the mismatch control vps15cont-mo with the zVps15-GFP reporter did not reduce the GFP expression as compared to controls injected with the zVps15- GFP plasmid. Embryos are 20 hpf old. E picture of WT and morphant zebrafish showing kidney cysts in the morphant (black arrow) but not the WT. F. vps15Δ yeast cells were transformed with a control empty plasmid (ctrl), an expression (CEN) or overexpression (2μ) plasmid bearing wild-type hVPS15 or mutant hVPS15-R998Q and a western blot against hVPS15 was performed. G. Wild-type (WT) and vps15Δ yeast cells transformed with a control (ctrl), hVPS15 or hVPS15-R998Q expression plasmid (CEN) were labeled with a soluble dye (CMAC) staining the lumen of the vacuole. White arrows show the additional CMAC positive compartment (CPC) in vps15Δ yeast cells. H. Drop test assays of wild-type WT and vps15Δ yeast cells expressing (CEN) or overexpressing (2μ) hVPS15 or hVPS15- R998Q. Plates were incubated at 30°C. Yeast strains transformed with an empty plasmid (ctrl) were used as controls.
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