Figure 3. C/EBP expression in macrophages with and with- out lymphocyte contact. (A) EMSA of whole cell THP-1 cell extracts with the HIV-1–LTR NRE that contains a C/EBP binding site. THP-1 cells after PMA differentiation (lane 1) and the same extract with excess- unlabeled NRE probe (lane 2). IFN- –treated THP-1 mac- rophages (lane 3) followed by coculture with activated lym- phocytes for 1 (lane 5) and 2 d (lane 7). IFN- –treated mac- rophages (lane 9) and supershift- ing with antibody to C/EBP (lane 10). (B) A Western blot of whole cell extracts of IFN- – treated THP-1 macrophages 1 (lane 1), 2 (lane 2), and 4 d (lane 3) after coculture of Con A–stimulated allogenic lympho- cytes. Separation of Con A–acti- vated lymphocytes from IFN- – treated THP-1 macrophages by a 0.4- m filter for 1 (lane 4), 2 (lane 5), and 4 d (lane 6). (C) A Western blot of TNP-1 macrophage before (lane 1) and 2 d after the addition of purified CD4 lymphocytes (lane 2). A nuclear extract before (lane 3) and 2 d after the addition of purified CD8 lymphocytes (lane 4). (D) NP-40 whole cell extracts made without (lane 1) and with (lane 2) calpain inhibi- tor. Ca2 is added to the extract without (lane 3) and with (lane 4) calpain inhibitor. Nuclear extracts made with NP-40 without (lane 5) and with (lane 6) calpain inhibitor. Cytoplasmic extracts without (lane 7) and with (lane 8) calpain inhibitor. All experiments were repeated three or more times with similar results. Figure 4. C/EBP expres- sion in THP-1 macrophages af- ter addition of antibodies. (A) Whole cell extract of IFN- – treated THP-1 macrophages 2 d after goat IgG without (lane 1) or with (lane 2) protein A/G agarose. Affinity-purified goat IgG specific for CD40, VCAM, and B7-1 was added without (lane 3) and with (lane 4) pro- tein A/G agarose beads for 2 d. Antibodies for B7-2 are substi- tuted for antibodies of B7-1 without (lane 5) and with (lane 6) protein A/G beads. (B) C/EBP Western of nuclear extract of IFN- –treated THP-1 mac- rophage (lane 1); 2 d after addi- tion of antibodies to VCAN and B7-1 without (lane 2) and with (lane 3) protein A/G beads; or antibodies to CD40 and VCAM (lanes 4 and 5); or antibodies to CD40 and B7-1 (lanes 6 and 7). (C) EMSA with the HIV-1–LTR NRE 2 d after combination of antibodies IgG specific for CD40, VCAM, and B7-1 without and with protein A/G beads (lanes 1 and 2). Antibodies for B7-2 are substituted for antibodies of B7-1 (lanes 3 and 4). Single antibodies to CD40, VCAM, B7-1, or B7-2 with and without protein A/G beads (lanes 5–13). All experiments were done at least three times with similar results. (D) Coculture of IFN- –treated THP-1 macrophages with Con A activate allogenic lymphocytes. C/EBP Western of whole cell extracts with the addition of blocking antibodies to lymphocyte expressed CD 40 ligand (CD 154), VLA-4 (CD 49 ), and CD 28 (lane 1). Lymphocyte addition without added antibodies (lane 2). Whole cell extracts of coculture experiments AM. Blocking added (lane 3) and no antibodies added (lane 4). 500 Contact and Cytokines Are Needed for Maximal HIV Replication