Figure 4
CnB stimulates phosphorylation of STAT1, and combination treatment with CnB and IFN-γ markedly enhances phosphorylation at Ser727 but not at Tyr701. (a) Western blotting detection of time-dependent phosphorylation of STAT1. RAW264.7 cells were treated with 20 μg/ml CnB for 0-300 min. (b) Proteinase K-digested CnB loses the ability to induce phosphorylation of STAT1 at Ser727. Cells were incubated with CnB (1–40 μg/ml) or proteinase K-digested CnB (1–40 μg/ml) for 25 min. (c) CnB pretreatment enhances IFN-γ-induced phosphorylation of STAT1 at Ser727 but not at Tyr701 (time course). Cells were preincubated with or without CnB (20 μg/ml) for 20 min, followed by incubation with IFN-γ (1 ng/ml) for 3–25 min. (d) Combined treatment with CnB and IFN-γ synergistically promotes STAT1 phosphorylation at Ser727 but not at Tyr701 (dose course). Duration of treatments, 25 min. Phosphorylation levels of STAT1 were quantified by densitometry and normalized to total STAT1 expression (Supplementary Figure S1)
