Figure 1.

Rho-Kinase mediates transforming growth factor β (TGF-β)-induced Jag1 expression in podocytes. Differentiated E11 podocytes were stimulated with TGF-β (5 ng/mL) for the indicated durations (A,B). (A) RNA was extracted, and Jag1 mRNA was analyzed by real-time quantitative PCR, with GAPDH mRNA as the internal standard; (B) The amount of Jag1 protein in cell lysates from podocytes was determined by Western blotting; (C) Cell lysates were collected from E11 podocytes stimulated with TGF-β (5 ng/mL) for 1 min. RhoA activity was determined by G-LISA RhoA assay; (D) Podocytes were pretreated with Y-27632 (10 µM) and then stimulated with TGF-β (5 ng/mL) for 1 h. Rho-kinase activity was measured as described in the Materials and methods section; (E) E11 podocytes were pretreated with Y-27632 (10 µM) before stimulation with TGF-β (5 ng/mL) for 8 h. Jag1 mRNA was analyzed by real-time quantitative PCR; (F) Podocytes were stimulated by TGF-β for 24 h after treatment with Y-27632. The protein expression of Jag1 was analyzed by Western blotting. A representative blot of three independent experiments is shown; (G) Podocytes stimulated with TGF-β (8 h) were treated with scramble control siRNA or Rho-kinase isoform specific siRNA and analyzed by real-time quantitative PCR. * p < 0.05 vs. control siRNA with TGF-β. The data are presented as means ± SD.