Figure 5
Stau1ΔNt88 interacts with HIV-1 Gag in live cells. (A) Schematic representation of the Stau1/CA-p2-NC-p1 BRET assay. (B) 293T cells were cotransfected with CMV-CA-p2-NC-p1-Rluc and Stau1-YFP expressors. 48 hours post-transfection, Stau155-YFP and Stau1ΔNt88-YFP contents in the cells were analyzed by Western blotting using anti (α)-GFP antibodies. (C) 293T cells were transfected with constant amounts of CA-p2-NC-p1-Rluc-expressing plasmid and increasing amounts of wild-type or mutated YFP-fused Stau1 expressors. Twenty four hours post-transfection, live transfected cells were used for CA-p2-NC-p1/Stau1 BRET assays. BRET ratios are plotted in function of their corresponding total YFP/Rluc ratio. n = 4. (D) BRET ratios were compared at identical total YFP/Rluc ratio and corrected by subtracting the background BRET ratio calculated for unfused YFP and CA-p2-NC-p1-Rluc co-expression. The corrected BRET ratio between CA-p2-NC-p1-Rluc and Stau155-YFP was arbitrarily set to 100%. n = 4. (E) 293T cells were co-transfected with Pr55Gag and wild-type or N-terminally truncated Stau1-Flag expressors. Twenty-four hours post-transfection, cell extracts were prepared and subjected to immunoprecipitation using anti-Flag antibodies. Cell lysates and immune complexes were analyzed by Western blotting using anti (α)-Flag and anti (α)-CA antibodies. Anti (α)-GAPDH antibodies were used as a loading control. n = 2.