3.4. ERK1/2 kinase activation activity of cp-hFGF7115-114
hFGF7 can trigger Ras-Raf-ERK1/2 MARK pathway signaling by direct interaction with the FGFR2b receptor, thereby further stimulating reactions such as epithelial cell proliferation and migration.40 Based on the previously reported works for mouse and human cell-based functional analysis of hFGF7,41, 42 we also evaluated whether the MARK signaling pathway was activated by recombinant cp-hFGF7115-114 using a mouse embryonic fibroblast NIH3T3 cell. NIH3T3 cells were treated with the purified cp-hFGF7115-114, and ERK1/2 phosphorylation in these cells was evaluated in a time- and dose-dependent manner.43 As shown in Figure 4A, cp-hFGF7115-114 (50 ng/mL) induced clear phosphorylation of ERK1/2, and its effect decreased in a time-dependent manner. In addition, cp-hFGF7115-114 also activated ERK1/2 phosphorylation in a dose-dependent manner that was similar to the commercially available wild type protein (Figure 4B). These results provided strong evidence that the CP did not profoundly change the biological activity of cp-hFGF7115-114 and thus retained a capability for phosphorylation of ERK1/2 in experimental cell lines.