Figure 1

Phosphorylation at SP sites in both the N- and C-terminal regions regulates xNgn2 activity. (A)³⁵S-labelled IVT xNgn2, or the indicated Serine-Proline to Alanine-Proline xNgn2 mutants, full phosphomutant (9S-AxNgn2), N-terminal region (NT-S-AxNgn2) or C-terminal region (CT-S-AxNgn2), were added to Xenopus laevis mitotic egg extracts and incubated at 21°C. Samples were taken at 0, 15, 30, 45, 60, 75, 90 and 120 mins and separated on 15% SDS-PAGE gels. Gels were analyzed by quantitative phosphorimaging analysis, calculating the average stabilization relative to wild-type xNgn2 within mitotic extract. Half-lives were calculated using first-order rate kinetics, and errors calculated using the Standard Error of the Mean (SEM). (B) Embryos were injected into 1 cell of 2 cells with the indicated amount of mRNA encoding GFP, xNgn2, CT-S-AxNgn2 or 9S-AxNgn2, injected side to the left. Embryos were fixed at stage 15 and subjected to in situ hybridization for neural ß-tubulin expression before being scored for increased neurogenesis on the injected side compared to the uninjected side on a scale of 0-3 [38]. The experiment was performed in duplicate (n = 17-37). (C) 1 cell-stage embryos were injected with 20 pg of mRNA encoding GFP, xNgn2, NT-S-AxNgn2, CT-S-AxNgn2 or 9S-AxNgn2, harvested at stage 15 and expression of xNeuroD analysed by qPCR (5 embryos per sample, n = 3).