Although the ClicG0472 and ClicEY04209 alleles were independently derived and backcrossed for several generations to normalize the genetic background, it was formally possible that the transposon insertions in Clic were not responsible for the blunted ethanol sensitivity in flies. To address this issue, we generated two revertants (ClicG0472.R4 and ClicG0472.R9) of the G0472 chromosome through precise excision of the transposon. Expression of Clic returned to normal in the two revertants (Figure 2B; one sample t tests; ClicG0472R.4, t(2)=0.0, p=1.00, n.s.; ClicG0472.R9, t(2)=1.22, p=0.348, n.s.; n=3) as did the ethanol sensitivity phenotype of ClicG0472 flies (Figure 3C; one-way ANOVA; genotype, F(3,44)=69.93, p<0.0001; *Bonferroni multiple comparison, Control w1118 t(22)=9.40, p<0.05; ClicG0472R.4 t(22)=7.68, p<0.05; ClicG0472R.9 t(25)=14.29, p<0.05; n=10–15). These data confirm that the G0472 transposon insertion disrupted Clic expression and blunted ethanol sensitivity.
