FIG. 6.

Effects of Anx2 disruption on Gag-mediated VLP production in multiple cell lines. (A to C) Jurkat cells stably transduced with lentiviruses expressing shRNA targeting Anx2 or control scrambled shRNA (Scr) were electroporated with pHXB2ΔBalD25S. Cells and VLPs were harvested at 48 h following electroporation, resolved by SDS-PAGE, and probed with anti-Gag (C) and anti-Anx2 antibodies by Western blotting. VLP production (A) was quantified from three independent experiments performed in duplicate and averaged. Levels of Anx2 were quantified (B); the inset shows a typical Western blot probed with anti-Anx2 and anti-actin antibodies. (D to F) 293T cells transiently transduced with lentiviruses expressing shRNA targeting Anx2 or control scrambled shRNA (Scr) were transfected with pHXB2ΔBalD25S. Cells and VLPs were harvested at 24 h following transfection, resolved by SDS-PAGE, and probed with anti-Gag (F) and anti-Anx2 antibodies by Western blotting. VLP production (D) was quantified from three independent experiments. Levels of Anx2 were quantified (E); the inset shows a typical Western blot probed with anti-Anx2 and anti-actin antibodies. (G to I) Wild-type (WT) MEFs (C57BL/6) and Anx2^−/− MEFs were transfected with pCMVGag. VLPs and cells were harvested at 24 h posttransfection, resolved by SDS-PAGE, and probed with anti-Gag (I) and anti-Anx2 antibodies by Western blotting. VLP production (G) was quantified from three independent experiments performed in duplicate. (H) Levels of Anx2 were analyzed by Western blotting with anti-Anx2 antibody, with anti-actin antibody serving as a loading control.