Fig. 5

Activation of ERK is regulated by Gas6.

(A) rhGas6 induced signaling pathway in HRMECs. Cells were incubated with rhGas6 (400 ng/ml) at various times, and cell lysates were subjected to western blotting with specific antibodies, as described in Materials and Methods. (B) Immunofluorescence staining for pERK1/2 and Hoechst staining was performed, as described in Materials and Methods. Representative images of p-ERK1/2 staining in rhGas6-treated HRMECs. (C, D) PD98059, SB203580, or U0126 was preincubated for 30 min, and HRMEC responses to rhGas6 or rhVEGF were determined by western blotting using specific antibodies. The experiment was repeated three times. (E) Phospho-ERK1/2 staining in the trunk of 26 hpf embryos [Ctrl-MO embryo (n = 7); gas6-MO injected embryo (n = 8); U0126-treated embryo (n = 8)] and quantitative analysis of zebrafish p-ERK1/2 expression by whole-mount immunostaining. The gas6-morphant and U0126 treated embryos showed reduction of p-ERK1/2 protein expression compared to controls in the zebrafish trunk. The experiment was repeated two times. **p<0.01 vs. Ctrl-MO.