Exposure of a cortical neuron to netrin induces the phosphorylation of focal adhesion kinase (FAK) at Tyr397 along the axon and within the growth cone (gc). Insert shows the control neuron (without treatment). A day-in-vitro 3 (DIV3) cultured cortical neuron was exposed to 100 ng/ml netrin for 30 minutes, fixed with 4% paraformaldehyde (PFA), then immunostained with anti-phospho-FAK (Tyr397) antibody (green) and Texas Red-X phalloidin (red). Scale bars, 20 µm; insert, 50 µm.
Cortical neurons were isolated and cultured from E17 embryo using a standard protocol as described in Lehnardt et al.,Proc Natl Acad Sci U S A, 100 (14): 8514-9. At DIV3, neurons were treated with 100 ng/ml netrin for 30 minutes, fixed with 4% PFA, then stained with anti-phospho-FAK (Tyr397) (Upstate) antibody and Texas Red-X phalloidin (red). Images were digitally acquired using a fluorescence microscope (Nikon Eclipse 660) equipped with Spot cooled CCD camera (Diagnostic Instruments).
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