Figure 4

The N-terminus of Stau1 is required for the modulation of Pr55^Gag multimerization in live cells. (A) Schematic representation of HA-tagged Stau1⁵⁵ expressors. Stau1 double-stranded RNA-binding domains (dsRBD) and tubulin-binding domain (TBD) are represented as grey and black boxes, respectively. (B) Schematic representation of the Pr55^Gag/Pr55^Gag BRET assay. This assay is used as a sensor of Pr55^Gag multimerization. 293T cells were transfected with constant amounts of pCMV-Pr55^Gag-Rluc and increasing amounts of pCMV-Pr55^Gag-YFP. A constant amount of a third plasmid expressing Stau1⁵⁵-HA₃ or Stau1^ΔNt88-HA₃ was included in the transfection procedure. Rluc activity as well as transmitted and total YFP activities was measured. BRET ratios were plotted in function of their corresponding total YFP/Rluc ratio which allows us to compare BRET ratios at the same relative expression levels of Pr55^Gag fusion proteins. This figure is representative of four independent experiments. (C) Cells corresponding to the four last points of each curve from Figure 4B were lysed. Cell lysates were analyzed by Western blotting using anti (α)-HA antibodies for their content in over-expressed Stau1 proteins. *: Non-specific labelling typically obtained with the anti-HA antibody. (D) BRET ratios were compared at comparable total YFP/Rluc ratio. The BRET ratio corresponding to the pr55^Gag fusions expressed alone was arbitrarily set to 1. The BRET induction levels were then determined and are shown in the graph. These results are representative of 4 experiments.