Fig. 3

RNF8 operates in the cytoplasm to suppress presynaptic differentiation in vivo. a Immunoblotting analyses of fractionated lysates of P8 rat cerebellum revealed abundant endogenous RNF8 in the cytoplasmic fraction in the cerebellum. b P4 rat pups were electroporated with an expression plasmid encoding an RNF8–GFP fusion protein together with a mCherry expression plasmid. After 8 days, rat pups were sacrificed and the cerebellum was subjected to immunohistochemistry using GFP and DsRed (mCherry) antibodies. RNF8 localizes predominantly in the cytoplasm. c P4 rat pups were electroporated with the control U6 plasmid or RNF8 RNAi plasmid together with an expression plasmid encoding NES(nuclear export signal)-RNF8Res fusion protein, NLS(nuclear localization signal)-RNF8Res fusion protein or the control vector and with the GFP expression plasmid and analyzed as in Fig. 1a. Expression of NES-RNF8Res, but not NLS-RNF8Res, reversed the RNF8 RNAi-induced phenotype of increased number of parallel fiber presynaptic boutons in the cerebellum in vivo (*p < 0.05, ANOVA followed by Fisher’s PLSD post hoc test, n = 3–4 rats). Scale bars = 10 μm