Fig. 14

Intracellular and surface expression of HIV-1 Env in the presence of gB. We examined the intracellular and surface expression of gB, HIV-1 Env, or both using COS-7 cells. For intracellular expression, cells grown on cover slips were transfected with vectors expressing gB and HIV-1. At 24 h, cells were fixed, permeabilized, reacted with a primary antibody against HIV-1 Env and gB for 16 h at 4C. The cells washed and reacted with appropriate secondary antibodies. Cells were counterstained with DAPI. For surface staining, cells on cover slips were transfected with vectors expressing gB, HIV-1 Env or both. At 24 h, cells were washed reacted with primary antibodies against gB and/or HIV-1 Env for 1 h at 4C, cells washed and fixed. The cells were then reacted with appropriate secondary antibodies and counterstained with DAPI. The cells were washed and the cover slips were mounted. The cells on coverslips were examined using a Leica TCS SPE confocal microscope using a 63 × objective with a 2 × digital zoom using the Leica Application Suite X (LAS X, LASX) software package. A 405 nm filter used to visualize the DAPI, 488 nm filter was used to visualize the gB, and a 594 nm filter to visualize HIV-1 Env staining. a–d Cells co-transfected with vectors expressing gB and HIV-1 Env that were fixed prior to staining. e–h Cells transfected with a vector expressing gB and reacted with the primary antibody against gB at 4C prior to fixation. i–l Cells transfected with vectors expressing HIV-1 Env and reacted with the primary antibody against HIV-1 Env at 4C prior to fixation. m–p Cells transfected with vectors expressing HIV-1 Env and gB, reacted with the primary antibodies against HIV-1 Env and gB at 4C prior to fixation. The results shown are representative of examining 50 transfected cells. a, e, i, m Visualization of gB staining. b, f, j, n Visualization of HIV-1 Env staining. c, g, k, o Visualization of DAPI staining. d, h, l, p Merge of the three panels to the left