Figure 7. PDGF signaling activates STAT1 in VSMCs.

(a) Tyrosine phosphorylation of STAT1, STAT3, and STAT5 in cultured VSMCs from PDGFRβ^Sm22D849V (β-D849V) mutant and Sm22α-Cre (Wt) control mice was determined by Western blot. As a positive control, VSMCs were treated with 10ng ml⁻¹ mouse IFN-γ for 30 min. (b) Phosphorylation of PDGFRβ and STAT1 in control VSMCs after serum starvation and treatment with 10 ng ml⁻¹ PDGF-BB for 15 minutes was determined by Western blot. PDGFRβ was immunoprecipitated from cell lysate and then phosphorylation was detected with anti-phosphotyrosine antibody. Phospho-STAT1 was detected in cell lysate with p-Y701 antibody. (c, d) PDGFRβ and STAT1 phosphorylation in PDGFRβ^Sm22D849V mutant and control VSMCs were analyzed by Western blotting after 16 hrs treatment with the PDGFR inhibitor 10 μM AG-1295. (e) STAT1 phosphorylation in PDGFRβ^Sm22D849V mutant and control VSMCs were analyzed by Western blotting after 16 hrs treatment with 10 μM AG-1295 followed by vehicle or 10ng ml⁻¹ mouse IFN-γ for 30 min. (f) Quantification of Western blots in (e) by densitometry shown as fold change over untreated Wt (lane 1). (g) STAT1 and PLCγ1 phosphorylation in PDGFRβ^Sm22D849V mutant and control VSMCs were analyzed by Western blotting after 16 hrs treatment with the JAK1/JAK2 inhibitor Ruxolitinib (10 μM) followed by 10ng ml⁻¹ mouse IFN-γ for 30 minutes or 10 ng ml⁻¹ PDGF-BB for 10 minutes. (h) Quantification of Western blots in (g) by densitometry shown as fold change over untreated Wt (lane 1). All data represent mean ± s.e.m. All experiments were performed at least three times; *P<0.01 by Student’s t-test