Fig. 5. BBG decreases the level of CaMKII-P at axonal growth cones. (A,B) Axon growth cones of hippocampal neurons (3 DIV) cultured in the presence or absence of BBG (5 µM) stained with antibodies against CaMKII/β (A) or CaMKII/β-P (B) (green) and phalloidin–Alexa-Fluor-594 (red). Scale bar: 25 µm. (C) The graph (bottom) represents the mean ± s.e.m. of the CaMKII/β-P fluorescence intensity at axon growth cones (relative units) in 150 neurons from three independent experiments (*P<0.05). Images (top) are 4 x magnifications of the axon growth cones of the hippocampal neurons shown in B and stained for CaMKII/β-P. (D) The levels of phosphorylated synapsin I (CaMKII substrate) in hippocampal neurons cultured in the presence of BBG (5 µM) for 30 or 60 minutes. Actin was used as a loading control. The graph represents the mean ± s.e.m. of the levels of phosphorylated synapsin I in three independent experiments (*P<0.05, paired t-test). (E) Hippocampal neurons cultured in the presence or absence of the CaMKII inhibitor KN-93 (1 µM). After 3 days in culture, the total axon length and the number of axon ramifications were quantified. The graphs represent the mean ± s.e.m. from three independent experiments (n=50; ****P<0.0001, paired t-test). Scale bar: 50 µm.