Figure 4
Coimmunoprecipitation assay between Nef and ACOT8 mutants.
(a) CHO cells were cotransfected with Nef-LAI and ACOT8 mutants as indicated in the panel. Nef-SF2 was used as negative control. Whole cell extracts were quantified and analysed with anti-HA or anti-ACOT8 antibodies, as indicated. α/β tubulin expression was used as a control. The Nef/ACOT8 mutants association was analysed by immunoprecipitation of the complexes with the anti-HA antibody. Western blot analysis of immunoprecipitated lysates, performed with the anti-ACOT8 antibody, shows that only the wild type ACOT8 and the p∆PAK mutant interact with Nef-LAI. CHO cells were cotransfected with either Nef-LAI (b) or Nef-SF2 (c) and increasing amounts of ACOT8. Similarly, they were cotransfected with increasing amount of either the p∆PAK (d) or the p∆PK mutant (e) and Nef-LAI. ACOT8 and Nef expression was analysed by Western blot. α/β tubulin expression was used as control. Nef and ACOT8 expression was calculated from the same membrane by densitometry analysis using the ImageJ software and normalized to α/β tubulin. Percentages relative either to the minimum amount of transfected ACOT8 construct, or to the Nef transfection, are indicated under each western blot image.
