Detection of HIV-1 Nef expression in HMØs after Ad-d1312–mediated transfection of nef cDNA. HMØs were transfected either with nef cDNA alone or cDNA conjugated with transferrin-poly(L-lysine) in the presence of a replication-deficient AD, dl312. HMØs were cultured in macrophage media in the presence of IL-3 (5 ng/mL) and GM-CSF (20 ng/mL). After 4 days in culture, cells were transfected and cultured in macrophage media containing FBS, IL-3, with/without GM-CSF for 24 hours. The cells were then labeled with 200 μCi/mL of ³⁵ S-labeled cysteine/methionine for 4 hours after starving them with cysteine/methionine-free media for 1 hour. Cells were then harvested, lysed, and immunoprecipitated with Nef antibody followed by SDS-PAGE and autoradiography. HMØs transfected with nef, AD, dl312, and lipofectamine in the presence of GM-CSF had the highest levels of Nef expression (lane 1). The absence of GM-CSF markedly reduced Nef expression (lane 2). The absence of Ad from the transfection resulted in minimal Nef expression in the presence of GM-CSF (lane 3) or in the absence of GM-CSF (lane 4).