Figure 1.
Molecular characterization of slob knock-out, knockdown and rescue flies. A, RT-PCR was done using total RNA from adult fly heads. Lane 1, DNA size markers (M). Compared with the parental line Kurs58 (lane 2), slob transcript is not detectable in the mutant lines mutK162 (lane 3) and mutIP1 (lane 4), but is present in the precise excision line WTP41 (lane 5). Predicted product size is 1.1 kb. B, Western blot analysis using lysates from adult fly heads collected at the same time of day and probed with polyclonal anti-Slob antibody. In contrast to line Kurs58 (lane 1), Slob57 protein is dramatically reduced/absent in the mutant lines mutK162 (lane 2) and mutIP1 (lane 3), and is recovered to at least wild-type levels in the precise excision line WTP41 (lane 4). C, Knockdown of Slob expression using transgenic RNAi. Western blot analysis conducted as described for B shows RNAi-induced knockdown of Slob57 expression. In contrast to the yw (lane 1), heterozygous Actin-GAL4 alone (Actin-GAL4/+, lane 2), and UAS-slob RNAi alone (UAS-slob RNAi/+, lane 3) control lysates, Slob57 expression is markedly reduced in lysates from flies carrying both Actin-GAL4 and UAS-slob RNAi (Actin-GAL4/UAS-slob RNAi, lane 4). D, Slob57 expression is restored to at least wild-type levels in rescue flies. Western blot conducted as described for B reveals that, in contrast to the lack of Slob expression in mutIP1 (lane 2), Slob57 levels in rescue flies (mutIP1 rescue, lane 3) are comparable to those seen in wild-type controls (WTP41, lane 1). Similar restoration of Slob57 expression is observed upon rescue of Slob-RNAi knockdown flies (data not shown).