FIG 2.
Expression of gp140 and Gag-Pol-Nef antigens by Western blot analysis. (A) Time course expression of HIV-1 antigens by Western blotting. Monolayers of BSC-40 cells were mock infected or infected at 5 PFU/cell with NYVAC-WT, NYVAC-C, NYVAC-gp140, or NYVAC-Gag-Pol-Nef. At different times postinfection (2, 6, 16, and 24 h), infected cells were collected, and cells extracts fractionated by 8% SDS-PAGE and analyzed by Western blotting using the polyclonal anti-gp120 antibody or the polyclonal anti-gag p24 serum to evaluate the expression of gp140 or Gag-Pol-Nef proteins, respectively. (B) Detection of HIV-1 proteins gp140 and Gag-Pol-Nef in the supernatant of infected cells by Western blotting. Monolayers of BSC-40 cells were infected at 5 PFU/cell with NYVAC-WT, NYVAC-gp140, or NYVAC-Gag-Pol-Nef. At different times postinfection (6, 18, and 24 h), cells were collected and supernatants (S) and cellular pellets (P) were processed as described in Materials and Methods, fractionated by 8% SDS-PAGE, and analyzed by Western blotting using the rabbit polyclonal anti-gp120 antibody or the rabbit polyclonal anti-gag p24 serum to evaluate the expression of gp140 or Gag-Pol-Nef protein, respectively. (C) Detection of the trimeric form of gp140 in the supernatant of infected cells by Western blotting. Monolayers of BSC-40 cells were infected at 2 PFU/cell with NYVAC-WT, NYVAC-C, or NYVAC-gp140. At different times postinfection (6 and 24 h), cells were collected and supernatants processed under nonreducing conditions as described in Materials and Methods, fractionated by 8% SDS-PAGE, and analyzed by Western blotting using the rabbit polyclonal anti-gp120 antibody.