Figure 4.

Prolonged activation of ERK MAPK is able to trigger the induction of robust circadian gene expression. NIH-3T3 cells were treated with 50% serum, 50 nm TPA, 200 μm OAG, 200 μm DOG, 30 nm EGF, 25 ng/ml FGF, or 30 ng/ml PDGF. (A) Whole-cell RNA was prepared at 70 min after treatment, and the relative levels of the mRNAs were determined by RNase protection assays. (B) Protein extracts prepared at the indicated times were subjected to immunoblotting with anti-ERK/MAPK antibody. EGF (C, 30 nm) or FGF (D, 25 ng/ml) was added to the medium (t = 0) and incubated for 2 hr as described. Relative levels of the mRNAs were determined by RNase protection assays. Three independent experiments gave similar results. (E) Signals obtained in the RNase protection assays shown in C and D (□, EGF; ●, FGF) for mPer2 and DBP mRNAs were quantified and normalized.