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Fig. 6. Dok-4 activates Rap1 that promotes neurite outgrowth in TGW cells. (A) Rap1 and Ras activation in TGW cells expressing Dok-4. Subconfluent TGW cells, TGW cells expressing Dok-4 wild-type or Y220F mutant, or Dok-4 knockdown cells were stimulated with GDNF (50 ng/ml) for the indicated times, thoroughly washed, and lysed. Lysates were clarified and incubated at 4°C with GST-RalGDS RBD (a) or GST-Raf-1 RBD (b) fusion proteins for 1 hour. Beads were washed, SDS sample buffer added, and the proteins subjected to western blot analysis for Rap1 (a) or Ras (b). (B) Rap1 induces neurite outgrowth downstream of Dok-4 in TGW cells. (a) TGW cells or Dok-4 knockdown cells were transfected with either GFP or GFP-RapV12 and incubated for 48 hours. (b) Mean of the longest neurite was determined for each culture from measurements of 50 neurons for three different experiments. Each data point represents the mean ± s.e.m.
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