Fusion with DUb had no effect on Alix known protein-protein interactions. (A) Schematic representation of the DUb-Alix fusion proteins. The active or inactive UL36 DUb catalytic domain was fused to Alix N-terminal region as depicted. (B) Effect of DUb fusion on Alix ubiquitination. 293T cells were transfected with Flag-Alix (500 ng), Flag-DUb-Alix (500 ng) or Flag-DUb*-Alix (500 ng) (lanes 1, 3, 5, 7, 9 and 11; respectively) or in combination with HA- Ub (lines 2, 4, 6, 8, 10 and 12; respectively). Immunocomplexes were analyzed by WB blot using the indicated antibodies. (C) DUb-Alix fusion proteins bind HIV-1 NCp6 and EIAV NCp9 proteins. GST, GST-NCp6 (right panel) or GST-NCp9 (left panel) fusion proteins were purified on glutathione beads and then incubated with lysates from 293T cells expressing 1.5 μg of Flag-Alix (lanes 2 and 8), Flag-DUb-Alix (lanes 4 and 10) or Flag-DUb*-Alix (lanes 6 and 12). Captured proteins and cell lysates were analyzed by WB blot using an anti-Flag antibody and GST fusion proteins visualized by Coomassie blue staining. (D) DUb-Alix fusion proteins retain binding to CHMP4B. 293T cells were co-transfected with HA-CHMP4B alone (2 μg) (control), or in combination with 1 μg of Flag-Alix (lane 2), Flag-AlixI212D (lane 3), Flag-DUb-Alix (lane 4) or Flag-DUb*-Alix (lane 5). Cell lysates were incubated with anti-Flag antibody-conjugated beads and both input and immunocomplexes analyzed by WB blot using the indicated antibodies.