Fig 4.

Suppression of endogenous GRB2 inhibits eMLV entry. A content-mixing assay was used to assess the impact of the reduction of GRB2 expression levels on virus entry. (A) The assay was adapted to 96-well plates, and the entry kinetics of eMLV particles were measured. In other assays, the entry signal reached a plateau after 120 min, and so a 90-min time point was chosen for subsequent assays. RLU, relative light units. (B) The impact of the GRB2-targeting siRNA was confirmed with cells used in the entry assay and showed a reduction in GRB2 levels but no change in either mCAT-1 or GAPDH levels in cells. (C) The impact of the indicated siRNA on eMLV entry into cells was then measured. Cells were transfected with siRNA and then challenged with eMLV gp85 (top)- or VSV-G (bottom)-pseudotyped, luciferase-containing MLV 72 h later. After 90 min, the luciferase activity in intact cells was measured by using luciferin-containing buffer lacking detergent. ∗, P < 0.05; ∗∗, P < 0.001 (determined by one-way ANOVA).