Figure 1

Assays for Nef oligomers by immunoprecipitation. (A) 293T cells were transfected to express untagged NA7Nef (lower molecular weight band, lane 2) and tagged NA7-HFNef and SF2-HFNef (higher molecular weight bands, lanes 3 and 4) singly and in the combinations of a tagged Nef with untagged NA7Nef. The membrane-associated protein fraction was prepared and Nef expression was determined by Western blot analysis (Input, lanes 1-6). Immunoprecipitations were also performed with anti-HA monoclonal antibody and were analyzed by SDS/PAGE and Western blot analysis (IP: α-HA, lanes 7-12). Antibody for the Western blots was sheep anti-HIV-1Nef. Arrows, untagged NA7Nef (U) present as hetero-oligomer and total tagged Nef (T). Upper T for SF2-HFNef and lower T for NA7-HFNef. (B) Same as in (A) with the corresponding cytoplasmic protein fractions. Note that in (A) the slightly lower molecular weight band present in the SF2-HFNef lanes running the same distance as the NA7-HFNef appears to have been proteolytically modified. In the soluble fractions additional minor proteolytic fragments are present.