Fig. 6.

RNF8 interacts with HERC2 and NEURL4 in the cerebellum and thereby suppresses parallel fiber presynaptic differentiation in vivo. a Lysates of Neuro2A mouse neuroblastoma cells constitutively expressing FLAG-RNF8 were subjected to immunoprecipitation followed by immunoblotting. Coimmunoprecipitation of FLAG-RNF8 with endogenous HERC2 and NEURL4. b The cytoplasmic fraction of lysates of P7 rat cerebellum was subjected to immunoprecipitation followed by immunoblotting. Endogenous coimmunoprecipitation of RNF8 and HERC2 in the cytoplasmic fraction of the cerebellum. c P4 rat pups were electroporated with the NEURL4 RNAi, HERC2 RNAi, or control U6 plasmid and analyzed as in Fig. 1a. Knockdown of NEURL4 and HERC2 increased the density of presynaptic parallel fiber boutons in the cerebellum in vivo (NEURL4 RNAi, ***p < 0.005, t test, n = 3–8 rats; HERC2 RNAi, ***p < 0.001, t test, n = 6–9 rats). d P4 rat pups were electroporated with the control U6 plasmid or RNF8 RNAi together with an RNF8Res mutant in which arginine 42 was replaced with alanine (RNF8ResR42A) or the control vector and with the GFP expression plasmid and analyzed as in Fig. 1a. Expression of RNF8ResR42A failed to reverse the RNF8 knockdown-induced presynaptic bouton phenotype in the cerebellum in vivo. Scale bars = 10 μm