Fig. 3

Validation of BirA*-Gag interactions by immunoprecipitation and Western blot. The Gag coding sequence was cloned into the pCI-Neo-Flag (Flag) (Life Technologies) between XhoI and NotI to express Flag-Gag. HeLa cells were transfected with either GFP or Gag-GFP (left panels) or Flag or Flag-Gag (right panel) for 24 h. Cell lysates were washed with PBS and collected using NP40 lysis buffer (50 mM Tris pH 7.8; 150 mM NaCl; 0.5 mM EDTA; 0.5 % Nonidet P-40) supplemented with Complete protease inhibitor (Roche). Cell lysates were quantified by the Bradford assay (Bio-Rad) and 1 mg of protein was immunoprecipitated with anti-MYC tag mAb-Magnetic beads for 2 h as described by the manufacturer (MLB). The bound complexes were analyzed by SDS-PAGE using anti-p24 (NIH, 183-H12-5C), anti-DDX17 (Abcam, ab24601) and anti-S6 Ribosomal Protein (Cell Signalling 54D2) antibodies