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Figure 5


Fig. 5. RhoA activity regulates p38 MAPK phosphorylation and kinase activity. (A) Western blot determination of active and total RhoA in lysates from C2 cells transiently transfected with empty vector (pcDNA), constitutively active RhoA (L63 RhoA) and dominant-negative RhoA (T19N RhoA). The band shifts observed for L63 and T19N RhoA are due to their Myc and HA tags (shown in B); thus the lower molecular weight bands show endogenous levels of RhoA. (B) Western blot determination of phospho-p38 (pp38), phospho-ERK1/2 (pERK1/2), total ERK1/2, Myc and HA tags and total p38 in whole cell lysates of C2 cells transfected and sampled as described in A. Phospho- and total p38 were semi-quantified by scanning densitometry and p38 phosphorylation normalised for total p38 calculated (lower panel); data are expressed as fold increases of p38 phosphorylation above pcDNA control levels; *P<0.05, ***P<0.001 compared with levels in the controls. (C) p38 MAP kinase activity in myoblasts transfected with pcDNA, L63 RhoA or T19N RhoA; total p38 levels were determined by Western blotting. (D) SRF promoter reporter activity in lysates extracted from C2 cells transiently transfected with pcDNA3, cN390{Delta}, L63 RhoA, WT RhoA or N19 RhoA (left panel) or treated with control extract or NcadFc (right panel); Western blot analysis of lysates shows transfected GFP and ß-actin levels.





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