BAG5 inhibits CHIP-mediated ubiquitinylation of α-syn <i>in vitro</i> and in cells.

<p>(A) <i>In vitro</i> ubiquitinylation assays were performed with increasing amounts of GST or GST-BAG5 as indicated. Ubiquitinylation was determined by Western blot using anti-α-syn, anti-CHIP, or anti-Hsp70 antibodies. GST fusion proteins were stained with Ponceau S. Similar results were found in three experiments. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, myc-CHIP, HA-Ub, and FLAG-BAG5 as shown. Ubiquitinylation of α-syn was detected using anti-HA antibodies (upper). The arrow indicates the monoubiquitinylated form of α-syn (UB-α-syn). Immunoprecipitation of equivalent amounts of α-syn was confirmed by probing with anti-α-syn antibodies (lower). The asterisk (*) indicates cross-reactivity of the secondary antibody with the immunoglobulin light chain. (C) Quantification by densitometric analysis of the UB-α-syn band with co-transfection of a control vector or FLAG-BAG5 from three independent experiments, one of which is represented in (B). Bars shown correspond to mean (±S.D.) gray value normalized to measures obtained for the control vector. *P<0.05, t-test versus control vector. (D) Protein expression levels of α-syn and CHIP with and without BAG5 overexpression was assessed by Western blot. Actin was used as a loading control.