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Figure 8


Fig. 8. p38 MAP kinase lies downstream of RhoA in the regulation of Igf2 expression. (A) Western blot detection of caveolin-3, phospho-ATF2, HA tag (MKK6EE expression), Flag tag (p38dn expression) and ß-actin in lysates from C2 cells that were transfected with control vector (pcDNA), constitutively active upstream activator of p38 MAPK, MKK6 (MKK6EE) and p38 dominant-negative (p38dn) constructs. (B) Northern blot analysis of Igf2 mRNA levels in C2 cells transiently transfected as in A; ethidium bromide staining of 18S rRNA is shown as a loading control. (C) Igf2 P3 promoter transactivation in C2 cells treated as in A but co-transfected with Igf2 P3 promoter and GFP. The lower panels additionally show western blot detection of MHC, HA tag (MKK6EE expression), flag tag (p38dn expression) and ß-actin. *P<0.05, ***P<0.001 (compared with an activation value of 1.0). (D) Northern blot analysis of Igf2 mRNA levels in C2 cells transiently transfected with control vector (pcDNA), cN390{Delta}, T19N RhoA alone or co-transfected with MKK6EE; GAPDH mRNA levels are shown as a loading control. (E) Igf2 P3 promoter-reporter activity in C2 cells treated as in C but co-transfected with Igf2 P3 promoter and GFP. Western immunoblot determination (lower panel) of HA tag (detects cN390{Delta}, T19N RhoA and MKK6EE expression), and total p38 (loading control). There is non-specific binding at ~50 kDa in the HA immunoblot which is present in all lanes. *P<0.05; **P<0.01; ***P<0.001 compared with an activation value of 1.0.





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