Removing PKA from microtubules using MAP2c constructs resulted in shorter neurites in dissociated hippocampal neurons.

<p>(A) E18 mouse hippocampal neurons were transfected with plasmid overexpressing MAP2c-ΔRII-EEE-EGFP (left column) or MAP2c-EEE-EGFP (right column) immediately after dissociation, incubated for 48 hours, and permeabilized with triton X-100 before formaldehyde fixation. Fixed neurons were stained with antibody against β-III-tubulin (top column) and antibody against PKA-RIIβ subunit (bottom). All scale bars equal 20 µm. (B) Quantification of microtubule-associated PKA in neurites. Error bars represent SD. All PKA signals were normalized to the PKA signal in untransfected neurons. (C) E18 mouse hippocampal neurons were transfected with plasmid overexpressing MAP2c-EEE-EGFP, MAP2c-ΔRII-EEE-EGFP, or EGFP immediately after dissociation and fixed after 48 hours. All images show signal from EGFP and were inverted to improve the visual presentation. All scale bars represent 50 µm. Quantification of total neurite length per neuron (D), average neurite length (E), and average neurite number per neuron (F) in transfected 2DIV hippocampal neurons. * p < 0.05, ** p < 0.01, one way ANOVA followed by Tukey’s post-hoc analysis. Error bars represent SEM from 3 independent repeats. More than 190 neurons were analyzed for each constructs.