Figure 5
CnB-induced STAT1 phosphorylation is dependent on the integrin αM–p38 signaling pathway. (a) CnB promotes phosphorylation of p38 in a time-dependent manner. RAW264.7 macrophages were incubated with 20 μg/ml CnB for 0–300 min, and then lysed for western blotting analysis. (b) The p38 inhibitor SB203580 markedly inhibits CnB-induced STAT1 Ser727 and Tyr701 phosphorylation. Cells were pretreated with SB203580 (1–50 μM) for 30 min followed by CnB/IFN-γ treatment for another 30 min. (c) CnB-induced p38 phosphorylation is blocked by integrin αM antibody. Antibody concentrations, 7.5 or 15 μg/ml; duration of antibody pretreatment, 45 min. (d) CnB-induced p38 phosphorylation is attenuated by integrin αM knockdown. Forty-eight hours after siRNAs transfection, cells were incubated with 5 μg/ml CnB for 25 min. (e) CnB-induced STAT1 phosphorylation is attenuated by integrin αM knockdown. Forty-eight hours after siRNAs transfection, cells were incubated with CnB (5 μg/ml) or IFN-γ (1 ng/ml) for 25 min. Phosphorylation levels of STAT1 and p38 were quantified by densitometry and normalized to total STAT1 or total p38 expression, respectively (Supplementary Figure S2)
