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. Author manuscript; available in PMC: 2017 Mar 9.
Published in final edited form as: Cell Host Microbe. 2016 Mar 9;19(3):336–348. doi: 10.1016/j.chom.2016.02.004

Figure 1.

Figure 1

Syntenin PDZs is ALIX Bro1 interacting partner. (A) In all experiments below, 293T cells were transfected with expression plasmids listed before lane numbers. Clear cell lysates were incubated with anti-HA antibody-conjugated beads (in E, anti-Flag antibody-conjugated beads were used) and both input and IP complexes were subsequently analyzed by SDS-PAGE and western blot (WB) using the indicated antibodies. (Left) Domain positions in syntenins are indicated. (Right) Syntenin binds ALIX through both Bro1 and V. Flag- Syn-1, Syn-2 or Syn-2β alone (lanes 1, 5 and 9), or with HA-ALIX (lanes 2, 6 and 10), HA-ALIX F676D (lanes 3, 7 and 11), or HA-Bro1 (lanes 4, 8 and 12). (B) Syntenin proteins bind ALIX in a LYPXnL motif-independent manner. Flag- Syn-1 or Syn-2 (lane 1, right and left panels, respectively), Flag-Syn-1 Y4,46,50F (lane 2, left panel), Flag-Syn-2 Y5F (lane 3, right panel), Flag–Syn-2 Y5,44F (lane 5, left panel) alone or with HA-ALIX (lanes 2, 4, and 6 right and left panels, respectively). (C) Endogenous syntenins co-associate with ALIX and Bro1. HA-ALIX (lane 2), HA-ALIX F676D (lane 3), or HA-Bro1 (lane 4). (D) Syn-2 PDZs are sufficient to interact with ALIX Bro1. Flag-Syn-2 PDZs (PDZ1+PDZ2) alone (lane 1), or with either HA-ALIX (lane 2), HA-ALIX F676D (lane 3), HA-Bro1 (lane 4) or control HA-APOBEC3G (A3G) (lane 5). (E) ALIX Bro1-Syn-2 PDZs optimal interaction requires the entire PDZ tandem. Flag-Syn-2 PDZs (1/2) (lane 1), PDZ1 (lane 3), (PDZ2) (lane 5) alone or with HA-ALIX Bro1 (lanes 2, 4 and 6). (F) ALIX Bro1-Syn-2 interactions are PI(4,5)P2-independent. Flag-Syn-2 (lane 1), Flag-Syn-2 mutPDZ1 (K113/K167A) (lane 3) or Flag-Syn-2 mutPDZ2 (K197/K244A) (lane 5) alone or with WT HA-Bro1 (lanes 2, 4, 6). (G) Drawing of the interactions between Syn-2 and ALIX. The red arrow marks the interaction identified in this study. See also S1 and S2.