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Figure 2. LPAR2 phosphorylation is required for in vivo NC migration. (A) Western blot of whole LPAR2 with Phos-tag PAGE showing that the phosphorylated forms of LPAR2 (Ph-LPAR2) become predominant at the onset of NC migration (stage 19). nonPh-LPAR2, nonphosphorylated LPAR2; α-Tub, α-Tubulin. (B) Normalized ratio of PhoshoLPAR2 and Phospho+nonphosphoLPAR2. The experiment was done four times. A representative gel and a representative graph showing the ratio of Phospho-LPAR2/Total LPAR2 are depicted in A and B, respectively. (C) Western blot of control (LPAR2) or point mutation S324A LPAR2 (LPAR2SA) cytoplasmic tail with Phos-tag PAGE. Alkaline phosphatase (+AP) treatment allows visualization of the non-phosphorylated bands. (D) Embryos injected with LPAR2MO or coinjected with LPAR2MO and 5 mismatch (5 mm) LPAR2 mRNA or 5 mmLPAR2SA mRNA. A mixture of FoxD3 and Dlx2 probes (which labels premigratory as well as migratory NC cells) was used to visualize the whole NC population. Asterisks indicate eyes. (E) Quantification of NC migration from embryos shown in D (18 embryos were analyzed). One-way ANOVA, P = 0.0025. Individual comparisons: *, P < 0.05. Error bars indicate SD. |
