CHIP mediates ubiquitinylation of α-syn in cells.
(A) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with HA-Ub, syn-luc1, and control vector or myc-CHIP as indicated. Immunoprecipitates were sequentially probed with anti-HA (upper) and anti-α-syn (middle) antibodies. Five percent of lysates used for immunoprecipitation was loaded as input and probed with anti-myc or anti-α-syn antibodies (lower). The middle band represents monoubiquitinylated α-syn (UB-α-syn). The asterisks (*) indicate immunoprecipitated bands that remain detectable by the anti-HA antibody following the substitution of all lysines within the α-syn sequence (see (B)). Molecular weight markers are indicated on left in kDa. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, synKR-luc1, HA-Ub, and myc-CHIP as indicated. Immunoprecipitated proteins were probed with anti-α-syn antibodies (H3C) which recognizes both syn-luc1 and synKR-luc1. The asterisks (*) correspond with the same bands indicated as such in (A). (C) Densitometric quantification of the band representing monoubiquitinylated α-syn when co-transfected with a control vector or myc-CHIP was performed from three independent experiments, one of which is represented in (A). Bars correspond to mean (± S.D.) gray value normalized to measures obtained for co-transfection of syn-luc1 and HA-Ub with control vector. *P<0.05, t-test versus control vector. (D) Proteins immunoprecipitated with anti-luc were probed with anti-α-syn antibodies (Syn-1) with a short or long exposure to film. The asterisks (*) correspond with the same bands seen in (A) and (B). (E) Immunoprecipitations with anti-α-syn were performed from lysates of H4 cells transfected with syn-luc1 and HA-Ub with control vector or myc-CHIP. Immunoprecipitated proteins were sequentially probed with anti-HA (upper) and anti-luc (lower) antibodies. The asterisks (*) correspond to the bands as indicated in (A), (B), and (D). Results are representative of three experiments.