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. Author manuscript; available in PMC: 2012 Oct 12.
Published in final edited form as: Structure. 2011 Sep 1;19(10):1485–1495. doi: 10.1016/j.str.2011.07.016

Figure 1.

Figure 1

(A) The Bro1 domains of Alix, HD-PTP and Brox bind HIV-1 NC domain. GST, GST-NCp6, or GST-NC proteins were expressed in E. coli, captured on Glutathione conjugated beads and subsequently incubated with lysates from 293T cells expressing either the HA-tagged version of the Bro1 domain of Alix (HA-Bro1, left panel), HD-PTP (HD-Bro1, central panel) or Brox (Brox, right panel). Immunoprecipitation complexes and input cell lysates were analyzed by SDS-PAGE and western blotting using anti-HA antibody and the expression of GST fusion proteins was visualized by Coomassie blue staining. (B) The Bro1 domains of HD-PTP and Brox do not promote HIV-1 release. Over-expression of both full length Alix and its isolated Bro1 domain rescue budding defects of HIV-1 PTAP- (lane 6) and PTAP-/YP- (lane 2) mutant viruses, respectively. 293T cells were transfected with pNL4-3 PTAP-/YP- proviral DNA alone (lane 1), with HA-Alix Bro1 (lane 2), HA-HD-Bro1 (lane 3), or HA-Brox, or with pNL4-3 PTAP- proviral DNA alone (lane 5), with HA-Alix (lane 6), HA-HD-PTP (lane7) or HA-Brox (lane 8). Cells and viruses were collected 24 hours post-transfection and their protein content was analyzed by SDS-PAGE and western blot using the indicated antibodies.