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Fig. 1. Knockdown of Dok-4 suppresses GDNF-dependent neurite outgrowth in TGW neuroblastoma cells. (A) (a) Expression of Dok-4 in TGW cells. Lysates from human cell lines were analyzed by western blotting with anti-Dok-4 polyclonal antibody under reducing conditions. HEK (human embryonic kidney), HeLa (human cervical adenocarcinoma), TGW (human neuroblastoma), SK-N-SH (human neuroblastoma), SK-N-MC (human primitive neuroectodermal tumor) and TT (human medullary thyroid carcinoma) cell lines were used. Lysate from HEK293 cells transfected with Dok-4 cDNA was used as a positive control. (b) Depletion of Dok-4 in TGW cells by siRNA. Total cell extracts from control siRNA- and Dok-4 siRNA-transfected TGW cells were subjected to western blot analysis with anti-Dok-4 and anti-Shc antibodies. (B) (a) TGW cells were transfected with control (left panel) or Dok-4 (right panel) siRNA, fixed 72 hours after transfection, and then stained with anti-Dok-4 antibody. Arrowheads denote the localization of Dok-4 to the plasma membrane. (b) TGW cells were serum-starved, incubated with or without GDNF (50 ng/ml) for 48 hours, and then stained with anti-Dok-4 antibody. Boxes 1 and 2 denote a shaft and a tip of an extended neurite which was positively stained with anti-Dok-4 antibody, respectively. (C) Depletion of Dok-4 attenuates neurite outgrowth by TGW cells in response to GDNF. (a) The target sequence of Dok-4 siRNA and the nucleotide substitutions used to generate siRNA-resistant (siRNAr) Dok-4 are indicated. (b) HEK293 cells were transfected with control or Dok-4 siRNA together with Dok4-V5 or siRNAr-Dok4-V5. Dok-4 expression was monitored by western blot analysis. (c) TGW cells transfected with siRNA were serum-starved, incubated with or without GDNF for 48 hours and then visualized by microscopy. TGW cells transfected with both siRNA and siRNAr-Dok4-V5 were also analyzed. (d) Mean of the longest neurite was determined for each culture from measurements of 100 neurons for three different experiments. Each data point represents the mean ± s.e.m. (*P<0.05).
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