Figure 2

The NC zinc fingers mediate Stau1/Pr55^Gag interaction. (A) Top: schematic representation of the Stau1/NC-p1 BRET assay. Bottom: 293T cells were transfected with constant amounts of pCMV-Stau1⁵⁵-Rluc and increasing amounts of wild type or mutated NC-p1-YFP expressors. 48 hours post-transfection, BRET ratios were determined and plotted in function of their corresponding total YFP/Rluc ratio which allows us to compare BRET ratios at the same relative expression levels of fusion proteins. This figure is representative of four independent experiments. (B) BRET ratios were compared at identical total YFP/Rluc ratio and corrected by subtracting the background BRET ratio calculated for unfused YFP and Stau1⁵⁵-Rluc co-expression (see Methods). The corrected BRET ratio for Stau1⁵⁵-Rluc and wild type NC-p1-YFP coexpression was arbitrarily set to 100%. These results are representative of four independent experiments. (C) 293T cells were transfected with Pr55^Gag-YFP or Pr55^{Gag C15-49S}-YFP expressors. Twenty hours post-transfection, lysates were analyzed by Western blotting using anti-GFP antibodies. (D) Following Stau1⁵⁵-Flag and wild-type or mutated Pr55^Gag-YFP co-expression, 293T cell lysates were submitted to immunoprecipitation using anti-Flag antibodies. Immune complexes were analyzed for their content of YFP-fused proteins and Stau1-Flag using anti (α)-GFP and anti (α)-Flag antibodies, respectively. Anti (α)-GAPDH antibodies were used as loading controls. This figure is representative of four independent experiments.