The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling
Figure 5
ROCK activity influences NOD1 signaling.
(A) HEK293T cells were transfected with CTRL or siRNA specific for the indicated target and incubated for 48 h. Subsequently, cells were transfected with the NF-κB luciferase reporter and NOD1 and stimulated with TriDAP (filled bars). After 16 h, cells were lysed, luciferase activation was determined and normalized with the β-galactosidase values (nRLU). Values are mean + S.D. (n = 3). Knock-down of the respective target is shown by immunoblot in the right panel. (B) Effect of the expression of constitutively active (CA) and dominant negative (DN) forms of ROCK1 on NOD1-mediated NF-κB activity measured by luciferase reporter assays in HEK293T cells. Values are mean + S.D. (n = 3). (C) NOD1-induced NF-κB activity measured by luciferase reporter assays in HEK293T cells for 6 h with the ROCK inhibitors Y-27632 and Glycyl-H1152. Values are mean + S.D. (n = 3). (D) THP1 cells were treated with the indicated amount of ROCK inhibitor Glycyl-H1152 for 6 h and TriDAP-induced IL-8 secretion was determined by ELISA. Mean + S.D. (n = 3) is shown. Cells were lysed and p-cofilin was detected by Western Blot analysis using a specific antibody (lower panel). Detection of GAPDH served as loading control. (E) Same experimental setting as (D), using HeLa cells stimulated with 10 µg/ml TriDAP. Mean + S.D. is shown. * p<0.05, ** p>0.005 compared to Ctrl (see also Figure S8).