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Figure 4 | Retrovirology

Figure 4

From: The host protein Staufen1 interacts with the Pr55Gagzinc fingers and regulates HIV-1 assembly via its N-terminus

Figure 4

The N-terminus of Stau1 is required for the modulation of Pr55Gag 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 Pr55Gag/Pr55Gag BRET assay. This assay is used as a sensor of Pr55Gag multimerization. 293T cells were transfected with constant amounts of pCMV-Pr55Gag-Rluc and increasing amounts of pCMV-Pr55Gag-YFP. A constant amount of a third plasmid expressing Stau155-HA3 or Stau1ΔNt88-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 Pr55Gag 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 pr55Gag 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.

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