FIG. 3.
GM-CSF-induced DNA binding of STAT5A is inhibited in HIV-1-infected MDM. Monocytes were cultured for 5 days prior to infection with HIV-1Ba-L (+) or mock infection (−). After 7 days, uninfected and HIV-1-infected MDM were incubated with 10 ng of GM-CSF per ml for 0 or 15 min and lysed for nuclear extraction. (A) Nuclear lysates were used in EMSA with 32P-end-labeled oligonucleotide containing the STAT response element of the FcγR1 promoter. Where indicated, extract was preincubated with an anti-STAT5A antibody (lanes 5 and 6), a nonspecific rabbit antibody (+) (lane 4), or unlabeled competing (cold comp.) probe (+) (lane 3) for 30 min at 4°C. Extract was preincubated with one of two different anti-STAT5A antibodies in lanes 5 and 6: antibody 1 was a rabbit anti-STAT5A antibody (α-STAT5A Ab) (Santa Cruz Biotechnology) (lane 5), and antibody 2 was a rabbit anti-STAT5A antibody (a gift from Lothar Hennighausen) (lane 6). The gel was dried, and the protein-DNA complexes visualized via autoradiography. IgG; immunoglobulin G. (B) The supernatant was removed from each culture of MDM prior to stimulation with GM-CSF, and the level of HIV-1 infection was assessed by RT assay. RT activity is given as counts per minute and represents activity in 1 μl of culture supernatant. Results are representative of MDM prepared from two donors.