Figure 6

Stau1^ΔNt88-HA ₃ does not affect the multimerization of membrane-associated Pr55^Gag. (A) Schematic representation of the experimental procedure. 293T cells were transfected with Pr55^Gag-Rluc, Pr55^Gag-YFP and Stau1-HA₃ expressors. 48 hours post-transfection, cytoplasmic extracts were prepared and subjected to the membrane flotation assay. (B) Pr55^Gag/Pr55^Gag BRET ratio was determined in each collected fraction. BRET ratios in fractions 1, 3, 4, 5 and 6 were omitted because Rluc activity in these fractions was too low to provide a reliable BRET ratio. (C) Schematic representation of the experimental procedure. 293T cells were transfected with HxBRU PR-provirus and Stau1-HA₃ expressors. 24 hours post-transfection, cytoplasmic extracts were prepared and subjected to the membrane flotation assay. Membrane fractions were collected and treated with 0.5% Triton X-100 during 5 minutes at room temperature in order to solubilize membranes. Resulting samples were subjected to ultracentrifugation at 100,000 × g during 1 hour. (D) Resulting supernatants (S100; S) and pellets (P100; P) were analyzed by Western blotting using anti (α)-CA, anti (α)-Na-K ATPase and anti (α)-HA antibodies. *: Non-specific labelling typically obtained with the anti-HA antibody.