Fig. 8

Mutations in the N-terminal domain of SIP decrease the ubiquitination of exon 1 mHTT. a Immunoprecipitation (IP) of ubiquitinated exon 1 mHTT in the presence of SIP and its dimerization mutants detected by Western blot (WB). Immunoprecipitation was performed in extracts from cells that were co-transfected with pEntry and e1HTT-72Q-RFP plasmids, wt SIP and e1HTT-72Q-RFP plasmids, SIP K21W and e1HTT-72Q-RFP plasmids, or SIP T30R_S33E and e1HTT-72Q-RFP plasmids. Immunoprecipitation was performed using anti-HTT N-terminal antibody (HTT) and analyzed for the presence of exon 1 mHTT ubiquitination (e1HTT-72Q-Ub or e1HTT-72Q-polyUb) with the application of an antibody that recognized ubiquitin (Ub; clone P4D1). The detection of co-precipitated exon 1 mHTT (e1HTT-72Q) using anti-HTT N-terminal antibody is shown in the control IP. Ab, corresponding antibody subjected to immunoprecipitation; pE, control pEntry plasmid; SIP wt, wildtype SIP; SIP K21W, mutant SIP K21W; SIP T30R_S33E, mutant SIP T30R_S33E; Total, total protein extract; ►, co-precipitating exon 1 mHTT or its ubiquitinated forms. IgG, heavy chain of antibody; M, protein marker in kilodaltons (kDa). As controls in panel a, total protein extracts were analyzed by WB for the electrophoretic mobility of e1HTT-72Q protein that was detected by N-terminal HTT antibody. SIP was detected by anti-SIP antibody. The loading control, GAPDH, was detected by anti-GAPDH antibody. b Densitometry of exon 1 mHTT ubiquitination bands in the presence of SIP and its dimerization mutants, detected by IP was calibrated based on the intensity of immunoprecipitated e1HTT-72Q in control IP and presented as fold changes. b Note that despite equal amounts of GAPDH in the total cell extracts there is less mHTT and less mHTT binds to the resin in the IP in the presence of wt SIP compared to control conditions and mHTT levels are elevated in the presence of SIP mutants. Therefore, the ubiquitination results from IP were normalized to the amount of mHTT that bound to the resin. The results are expressed as mean ± SEM. *p < 0.05, ***p < 0.001. ns, not significant. The results were obtained from five independent HEK293T culture preparations