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.