Figure 2. MEE treatment of ALIX_nPRD inhibits its interaction with ALIX_Bro1.

(A) Schematic illustration of the regions of ALIX that comprise the three structural domains and the ALIX fragments used in this study. (B) GST-ALIX_1-746 was phosphorylated with IOE, MEE, or MEE plus CIP, and end products were immunoprecipitated with the indicated antibodies, followed by IB with α-GST. (C) GST or GST-ALIX_1-746 was phosphorylated with IOE or MEE, and then absorbed onto GSH beads. The washed beads were used to pull down FLAG-CHMP4b in IE. IB of input and bound proteins with the indicated antibodies. (D) Left: experimental flowchart. Middle: IB of input myc-ALIX_nPRD; the short line and the asterisk indicate the start and shift positions of myc-ALIX_nPRD, respectively. Right: IB of bound proteins. (E) Phosphorylation of GST or GST-ALIX_Bro1 with IOE, MEE, or MEE plus CIP, followed by IB of washed proteins with αphosphotyrosine (p-Tyr). (F) Washed GST or GST-ALIX_Bro1 proteins in (E) were tested for interaction with myc-ALIX_nPRD by GST pull-down (n = 3 ± SD).