Analysis of MAPK activation in response to MNNG.
Whole cell extracts (WCE) were prepared as detailed in “Material and methods” from C2C12 and NIH3T3 cells at the indicated time points after MNNG stimulation. (A) MNNG induces JNK activation in C2C12 (left) and in NIH3T3 (right) cells. (Top panel) JNK activity was determined by immunoprecipitation of JNK1 from WCE, followed by kinase assay using the substrate GST-cJun.1-79 (Bottom panel) Western blot showing total JNK1 amount present in each cell extract (30 μg/lane). (B) MNNG does not induce ERK activation in C2C12 cells. Cells were stimulated with either 70 μmol/L MNNG or 20% serum as indicated. (Top panel) ERK activity was determined by immunoprecipitation of ERK2 from WCE, followed by kinase assay using the substrate MBP. (Bottom panel) Western blot showing total ERK2 amount present in each cell extract (30 μg/lane). (C) MNNG induces phosphorylation of p38 in C2C12 cells. Cells were either treated with 70 μmol/L MNNG or irradiated with UV-C (254 nm, 30 J/m2) 70 μg WCE were resolved by SDS-PAGE and phospho-p38 was detected by Western blotting using an anti-phospho-p38 antibody.
