Figure 2

Western blot analysis of transfected HeLa-tat cell and precipitated viruses. HeLa-tat cells were transfected with the plasmids indicated using the non-liposomal transfection reagent. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Particles released into the culture supernatant were also clarified and filtered of cell debris and precipitated with Viraffinity (CPG) as recommended by the manufacturer. Denatured cell (A and B) and viral lysates (C) were then separated by SDS-PAGE, transferred onto a nitrocellulose membrane and detected with a rabbit anti-HIV glycoprotein (A), a pool of anti-CAp24 and anti-calnexin (B), and anti-CAp24 (C) antibodies. The positions of specific viral proteins are indicated to the left and the numbers to the right depict positions of molecular mass markers (in kDa). NT, a mock control; WT, wild type; and D51N, D51E, and D51Q are the three CAp24 mutants.