Figure 6 : ERK7 phosphorylates CapZIP.

From: ERK7 regulates ciliogenesis by phosphorylating the actin regulator CapZIP in cooperation with Dishevelled

Figure 6

(a) Immunoblotting analysis of the phosphorylation status of CapZIP in lysates from HEK293T cells transfected with the indicated combinations of expression vectors. (b) Cilia (green) on the GRP. Each mRNA (1.5 ng per cell) was injected with mem-mCherry (red) mRNA into the dorsal marginal region of two blastomeres at the four-cell stage. The lower graph shows the average length of cilia on the GRP (luciferase, n=30 from five dorsal explants; CapZIP WT, n=84 from five dorsal explants; CapZIP 2A, n=82 from six dorsal explants; CapZIP 6A, n=19 from three dorsal explants). Scale bars, 5 μm. Error bars represent the s.d. ***P<0.001 by the t-test. (c) In vitro kinase assays using purified ERK7 and CapZIP proteins. FLAG-CapZIP and FLAG-ERK7 were expressed separately in HEK293T cells, and purified as shown by Coomassie brilliant blue staining (right panel). (d) The indicated sets of MO (10 ng per cell), luciferase or CapZIP 6E mRNA (1 ng per cell) and Centrin2-EGFP mRNA (100 pg per cell) were injected into the ventral marginal region of one blastomere at the four-cell stage. Centrin2-EGFP was used as a tracer. The injected embryos were harvested at stage 35/36 and immunostained with anti-acetylated α-tubulin. The number of cilia in MCCs (n=100) from 10 embryos of two independent experiments for each condition was scored. Representative images of MCCs are shown for each condition. Scale bars, 5 μm. *P<0.05 by the Mann–Whitney U-test.