Figure 6

Downstream of tyrosine kinase/docking protein (Dok)6 is involved in cortex neuron neurite outgrowth through the neurotrophin 3 (NT-3)/tropomyosin-related kinase (Trk)C pathway. (a) Dok6 binds to the TrkC receptor in cortical neurons upon NT-3 stimulation. E18.5 mouse cortex neurons were cultured in empty DMEM for 24 h, then treated with or without 100 ng/ml NT-3 for 10 min. Cell lysates were immunoprecipitated by anti-Dok6 and were then detected by western blotting with anti-TrkC and anti-Dok6. (b) E18.5 mouse cortex primary cells were transfected with Dok6i and nonsense control constructs (green) separately; 12 h later, cultured cells were transferred into DMEM with/without 100 ng/ml NT-3 for another 72 h. Then immunofluorescence was performed with anti-microtubule-associated protein 2 (MAP2; red) and photographs of cells that were positive for both red and green fluorescence (white arrow) were taken. Panels a and b show the nonsense control group treated without or with 100 ng/ml NT-3; panels c and d show the Dok6i group treated without or with 100 ng/ml NT-3. Scale bars for panels a, c and d are 100 μm, panel b is 200 μm, and panels e-p are 30 μm. (c) Quantification of neurite outgrowth in cortex neurons. A total of 10 random fields of cells from each experimental group were photographed. Neurite length was quantified by measuring the longest neurite processes of cells that were positive for both green and red fluorescence by using NIS-Elements software. *P value < 0.05 by Student t test.