Fig. 5

Ubiquitination of N-terminal wt HTT and mHTT increases in the presence of SIP. Immunoprecipitations (IP) of ubiquitinated exon 1 wt HTT a or mHTT b in the presence of SIP detected by Western blot (WB). a Immunoprecipitation was performed in extracts from cells that were co-transfected with both control pEntry and e1HTT-25Q-RFP plasmids or both wt SIP and e1HTT-25Q-RFP plasmids. b Immunoprecipitation was performed in extracts from cells that were co-transfected with both control pEntry and e1HTT-72Q-RFP plasmids or both wt SIP and e1HTT-72Q-RFP plasmids. In a and b, IP was performed using anti-HTT N-terminal antibody (HTT) and analyzed for the presence of exon 1 HTT ubiquitination (e1HTT-25Q-Ub and e1HTT-25Q-polyUb or e1HTT-72Q-Ub and e1HTT-72Q-polyUb, respectively) with the application of an antibody that recognized ubiquitin (Ub; clone P4D1). In b, control IP presents the co-precipitation of exon 1 mHTT (e1HTT-72Q) using anti-HTT N-terminal antibody. ^#In the control IP experiments in panel a, bands at 55 kDa that corresponded to exon 1 of wt HTT fused with RFP tag (encoded by e1HTT-25Q-RFP) and IgG heavy chain could not be separated in 8% polyacrylamide gels. Ab, corresponding antibody subjected to IP; pEntry, control pEntry plasmid; SIP wt, wildtype SIP; Total, total protein extract; ►, co-precipitating exon 1 of wt HTT or mHTT or its ubiquitinated forms; IgG, heavy chain of the antibody; M, protein marker in kilodaltons (kDa). As controls in a, total protein extracts were analyzed by WB for the electrophoretic mobility of exon 1 wt and mHTT detected by N-terminal HTT antibody. SIP was detected by anti-SIP antibody and the loading control GAPDH was detected by anti-GAPDH antibody. c Level of ubiquitination in total protein extracts from HEK293T cells that were treated with different concentrations of the proteasome inhibitor MG132. d Densitometry of exon 1 mHTT ubiquitination (e1HTT-72Q-Ubiquitin) bands detected in the presence of wt SIP and control pLenti plasmid was calibrated based on the intensity of immunoprecipitated exon 1 mHTT (e1HTT-72Q) and presented as fold changes. 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. Therefore, the ubiquitination results from IP were normalized to the amount of HTT that bound to the resin. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01. ns, not significant. The results were obtained from at least three independent HEK293T culture preparations