Flies that lack both RDGC-S and RDGC-M isoforms exhibit RH1 hyperphosphorylation.

A, Heads of flies lacking RDGC-S (rdgCΔS), RDGC-S and RDGC-M (rdgCΔSM), and RDGC-L (rdgCΔL) as well as from control flies were subjected to Western blot analyses using an α-RDGC antibody. B, In order to confirm the epitope of the monoclonal 4C5 α-RH1 antibody, wild type, rh1CT S>A, rh1Δ356, and ninaE17 fly heads were subjected to Western blot analyses. The rh1CT S>A fly expresses RH1 in which the putative C-terminal phosphorylation sites were exchanged by Ala and the rh1Δ356 fly expresses a C-terminally truncated RH1. Western blots were probed with the 4C5 α-RH1 antibody (upper panel) and a polyclonal α-RH1 antibody (lower panel). C, Dark-reared flies of the indicated genotypes were illuminated for 1 h (white bars) or kept in the dark (black bar) and head extracts were incubated with (+) or without (-) ʎ-phosphatase (ʎ-PP). Head extracts were then subjected to Western blot analyses using monoclonal (4C5) and polyclonal α-RH1 antibodies as well as an α-RDGC antibody. D, Dark-reared flies of the indicated genotypes were illuminated for 1 h (white bars) or kept in the dark (black bars) and head extracts were subjected to Western blot analyses using monoclonal (4C5) and polyclonal α-RH1 antibodies as well as an α-RDGC antibody. E, rdgCΔS, rdgCΔSM, rdgCΔL, and control flies were reared in the dark and illuminated for one hour before fly heads were subjected to Western blot analysis. A representative blot (E) and the quantification of signals obtained with the 4C5 antibody (F) are shown. The normalized integrated 4C5 signal intensity was calculated by dividing the integrated pixel intensities of the bands obtained with the 4C5 antibody by those of corresponding bands obtained with the polyclonal α-RH1 antibody. Error bars in (F) show SD. The significance was calculated using ANOVA with Dunnett’s multiple comparison test. n.s., not significant; n.d., not detected; ** p ≤ 0.01. G, Eyes of the indicated flies were subjected to Western blot analyses using α-RDGC and α-tubulin antibodies. H, Signals from RDGC-S and -M protein variants or the RDGC-L protein isoforms were quantified and normalized by tubulin signals. N = 6–9, error bars show SEM.