FIGURE 6.

ROCK is involved in NF-κB activation. A, RAW264.7 cells were co-transfected with the NF-κB-luciferase reporter construct and the pCS2+-β-galactosidase plasmids. The transfected cells were pretreated with 10 μm Y27632 and 10 μm HA1077 for 1 h and then incubated with TGF-β1 for 1 h. Luciferase and β-galactosidase activities were measured. B, cells were pretreated with 10 μm Y27632 or 0.5 μm 5z-7-oxozeaenol (OXO) for 1 h and then with TGF-β1 for the indicated times. The phosphorylation of IκB, IKKα/β, and p65 was analyzed by Western blotting. The data represent the means ± S.E. of three independent experiments (*, p < 0.05; **, p < 0.01). C and D, GST-IKKβ (0.1 μg), ROCK (0.1 μg), with 10 mm MgCl₂ and 25 μm ATP were incubated for 30 min at 30 °C in the presence (D) or absence (C) of cell lysates (50 μg), and p-IKKα/β (Ser-176/177 and Ser-180/181) and phospho-myosin light chain phosphatase (p-MYPT) were identified with Western blotting. E, RAW264.7 cells were first pretreated with DMSO or 0.5 μm 5z-7-oxozeaenol dissolved in DMSO for 1 h and then incubated with TGF-β1 for 1 h. RhoA-GTP levels were determined using a GST-rhotekin-RBD pulldown assay. C, control; T, TGF-β1. The data represent the means ± S.E. of three independent experiments (*, p < 0.05; **, p < 0.01), and the means ± range of two independent experiment for p-IKK α/β at Ser-176/177.