GM-CSF induces an early and long-lasting activation of STAT5Δ, ERK2, and a later up-regulation of HIV expression in U1 cells. (A) STAT5Δ expression (upper panel) and activation (lower panel) was assessed by incubation with the same Ab indicated in Figure 1, and by Western blotting of WCE of U1 cells stimulated with GM-CSF and collected at the indicated times after stimulation. STAT5Δ activation was detected for at least 48 hours after cytokine stimulation. (B) ERK-2 activation persisting up to 36 h after GM-CSF stimulation of U1 cells was demonstrated by Western blotting, using either anti-ERK-2 (upper panel) or anti-phosphorylated-ERK (lower panel) Ab. (C) Opposite effects of PD98,059 and AG490 on GM-CSF induced HIV-1 expression in U1 cells. U1 cells were stimulated with GM-CSF (20 ng/mL) in the presence or absence of the ERK inhibitor PD98,059 (20 μM) or the JAK2/JAK3 inhibitor tyrphostin AG490 (200 μM). Culture supernatants were collected daily to measure RT activity production and accumulation. The results shown were obtained in a single experiment representative of three independently performed in duplicate cultures (mean ± standard deviation).