Fig. 6.

Molecular dissection of HD-PTP Bro1 binding. (A) His-tagged bacterially expressed Bro1 domains of Alix, HD-PTP or HD-PTP (L202D) or (L202D/I206D) mutants were incubated with GSH beads preloaded with GST-CHMP4B or control beads. After binding, samples were analyzed by Coomassie (Upper) or Western blot with anti-His (Lower). Arrows indicate positions of bound Alix and HD-PTP Bro1 domains. CHMP4B band and minor CHMP4B degradation products are present. (B) In vitro translated HD-PTP, the indicated mutants (Left), or the Bro1-V domain and indicated mutants (Right) were incubated with GSH beads preloaded with GST or GST-CHMP4B. Loading controls for GST-CHMP4B binding are shown below. Arrows indicate the position of full-length HD-PTP. (C) Data from binding translated constructs. Values for each mutant are expressed relative to the binding of either WT full-length HD-PTP or the WT Bro1-V construct, after subtracting binding to GST controls. Values are means of three experiments ± SD.