Fig. 5

RhoA participates in thrombin-stimulated IL-8/CXCL8 expression in A549 cells. A A549 cells were treated with 30 μM Rhosin before being stimulated for the next 24 h with thrombin (10 U/ml). The level of IL-8/CXCL8 release was verified (n = 4, mean ± SEM, *p < 0.05 vs thrombin-stimulated cells). B A549 cells were treated with thrombin (10 U/ml) for the indicated periods. A Western blot pulldown assay was performed to determine RhoA activity (RhoA-GTP) level. Total RhoA level was used as a loading control (n = 4, mean ± SEM, *p < 0.05 vs non-stimulated cells). C Membrane fraction of A549 cells was extracted after thrombin (10 U/ml) for the indicated periods. Immunoblotting was performed to determine the protein level of RhoA in the membrane fraction. E-cadherin was used as a plasma membrane marker (n = 4, mean ± SEM, *p < 0.05 vs non-stimulated cells). D A549 cells were transfected with control (con siRNA) or DCLK1 siRNA for 48 h and subsequently treated with 10 U/ml thrombin for 5 min. A Western blot pulldown assay was performed to determine RhoA activity (RhoA-GTP) level. Total RhoA level was used as a loading control (n = 3, mean ± SEM, *p < 0.05 vs thrombin-stimulated cells). E A549 cells were transfected with control (con siRNA) or DCLK1 siRNA for 48 h and subsequently treated with 10 U/ml thrombin for 5 min. Membrane fraction were immunoblotted to determine the expression level of RhoA. (n = 4, mean ± SEM, *p < 0.05 vs thrombin-stimulated cells)