FIGURE 6.

Neurons expressing a non-phosphorylatable γ-Pcdh exhibit increased dendrite arborization. A and B, representative cortical neuron micrographs from cultures transfected at low efficiency with an empty vector, A3-WT, A3-ΔC15, or A3-S/A plus GFP to allow for tracing. Cultures were treated at 5 DIV with the FAK inhibitor PF-228 (B) or vehicle alone (DMSO; A) and analyzed at 8 DIV. C, Sholl analysis curves demonstrate increased dendritic complexity of neurons expressing A3-S/A compared with A3-WT or empty vector. No decrease in arborization was observed in neurons expressing A3-ΔC15, suggesting that lipid binding of the γC domain is not critical to regulate FAK. All neurons treated with PF-228 (C, dotted lines) exhibit similarly high dendritic complexity regardless of transfection conditions. Expression of A3-S/A did not result in a further increase in arborization, consistent with its effect being to increase the level of FAK inhibition, which is maximized by PF-228 treatment. D, area under the curve graphs of Sholl analysis data in C. E, Western blots of lysate from neurons nucleofected with A3-WT or A3-S/A, probed with the indicated antibodies. F, FAK activation, measured as the normalized PFAK/FAK ratio, is significantly reduced in neurons expressing A3-S/A (left axis). This is not due to higher expression of this construct (right axis). The graph shows results from three separate cultures ± S.E., n = 54–60 neurons per condition. **, p < 0.01. The bar in A is 100 μm.