FIG. 7.

Effect of expression of Anx2 in trans on VLP production. (A and B) COS-1 cells were cotransfected with pHXB2ΔBalD25S and either empty vector (pCDNA3.1) or vector encoding Myc-His-tagged Anx2. Cells and VLPs were harvested at 24 h following transfection, resolved by SDS-PAGE, and probed with anti-Gag and anti-Anx2 antibodies. The upper band in the Anx2 blot represents the exogenous Myc-His-tagged Anx2, while the lower bands are endogenous Anx2 (A) Western blots of Gag and Anx2 expression. (B) VLP production was quantified; the graph represents three independent experiments. (C and D) 293T cells were cotransfected with pHXB2ΔBalD25S and either empty vector (pCDNA3.1) or vector encoding Myc-His-tagged Anx2 or Flag-tagged p11. Cells and VLPs were harvested at 24 h following transfection, resolved by SDS-PAGE and probed with anti-Gag, anti-Anx2, and anti-p11 antibodies. (C) Western blots of Gag, Anx2, and p11 expression. The upper band in the Anx2 blot represents the exogenous Myc-His-tagged Anx2, while the lower bands are endogenous Anx2. The upper band in the p11 blot represents exogenous Flag-tagged p11, and the lower band is endogenous p11 (D) VLP production was quantified; the graph represents three independent experiments. (E and F) 293T cells were transfected with pNL4.3 and either vector or Anx2. Cells and virus were harvested at 48 h posttransfection. Levels of Gag, actin, and Anx2 were determined by Western blotting (E), and p24 levels and infectivity (relative light units obtained in the luciferase assay per ng p24) (F) were determined as described in Materials and Methods. Experiments were performed two times in triplicate; results from a representative experiment are shown.