Fig 1: Validation of stimulus and poststimulus phospho-signalling.(A) Heat map of significantly regulated phosphorylation sites for syn1 (upper) and dyn1 (lower) from 20 mM and 76 mM KCl stimulated synaptosomes (left, n = 6) and a single 10-s time point of 76 mM KCl stimulated hippocampal neurons (right, “ns” means not significantly regulated, n = 3). The log2(stimulated intensity/control intensity) is shown using the same colour scale as Fig 2B. Note: phospho-S662 in syn1 was detected with differential regulation from 1a and 1b isoforms. The synaptosome data are the result of six independent experiments for each stimulation condition (20 mM and 76 mM KCl). The cultured hippocampal neuron data are from three independent experiments. (B) Representative western blots of syn1-pS603, syn1-pS62+S67, dyn1-pS774, and ß-actin loading control for 76 mM KCl depolarized and repolarized synaptosomes. Bar graphs of the densitometry of the western blots, after correction for loading, are shown (below). The intensities were normalized to the control (“Cont.”)/mock stimulation. The bar graphs show the mean and standard deviation of 3 (syn1 pS603), 4 (dyn1 S774), or 5 (syn1 S62+S67) independent experiments. Statistical significance was determined by one-way analysis of variance with Dunnett’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; in (B), “ns” means “not significant”. P = 0.0005 using Student t test to compare dyn1-pS774 intensity at 10 s versus control and P = 0.17 when adjusted for multiple comparisons (time points). The heat map rows in (A) for the sites examined in (B) are boxed. Underlying data for this figure can be found in S1 Data. (C) Domain structure of Syn1 using A-E domain naming [39] and Pfam evolutionarily conserved domains [34] with the activity-dependent phosphorylation site positions indicated. dyn1, dynamin 1; syn1, synapsin 1.