FIGURE 1.

Nef interacts with both Bro1 and V domain of Alix. A, schematic representation of N-terminal GST-tagged Alix domain constructs. Below each of the three domain structures of Alix, Bro1 domain, V domain (VD), and proline-rich domain (PRD), are listed the known interaction partners. B, in vitro analyses of interactions between Nef and Alix domains. Recombinant GST and GST fusion proteins were produced in E. coli and immobilized onto glutathione-Sepharose beads, as described under “Experimental Procedures.” HEK-293T Nef-strep-FLAG cells were cultivated with 1 μg/ml doxycycline to induce expression of Nef-strep-FLAG. Total cell lysates were prepared and incubated with immobilized GST, GST-AlixFL, and truncated Alix-GST proteins as follows: GST-Bro1, GST-VD, or GST-PRD. Binding of Nef to GST fusion proteins was analyzed by immunoblotting with anti-FLAG antibody (top). An aliquot of 10% input of recombinant proteins (Input) was subjected to SDS-PAGE and stained with Coomassie Blue (bottom). C, Nef binds to the N-terminal region of Bro1. Shown is a schematic representation of the C-terminal deletion mutants of Alix used for GST-pulldown analyses (top). Recombinant GST fusion proteins were produced in E. coli and immobilized as in B and incubated with total cell lysate of HEK cells expressing V5 epitope-tagged Nef (Nef-V5). Binding of Nef to GST fusion proteins was analyzed by immunoblotting with anti-V5 (left panel). An aliquot of 10% input of recombinant proteins (Input) was resolved by SDS-PAGE and stained with Coomassie Blue (middle and right panels). The molecular masses are indicated in kilodaltons.