Fig. 5.

Phosphorylation on multiple serine-proline (SP) sites has an additive effect on Ngn2 DNA binding. (A) EMSA analyses in Xenopus M extracts showing E box binding of normalised IVTs of xNgn2 or mNgn2 and additive phosphorylation site mutants of xNgn2 or mNgn2 as labelled. Graphs show quantitation of E box binding of phosphorylation site mutants by phosphorimaging, normalised to DNA binding of xNgn2 or mNgn2 as appropriate (see Fig. S1 in the supplementary material). (B) qPCR analysis of neural βIII-tubulin in P19 cells. P19 cells were transiently transfected with mNgn2 and additive phosphorylation site mutants thereof for 24 hours. Average fold increase (± s.e.m.) in βIII-tubulin mRNA expression normalised to housekeeping genes [β-actin (Actb) and Gapdh]. ^**, P≤0.05; ^***, P≤0.005.