Figure 1

Chemical crosslinking of TrkB on the cell surface. (A) CHO cells expressing GFP-TrkB were stimulated with (+) or without (-) 100 ng/ml of BDNF for 5 min at 37°C. The cells were lysed and subjected to Western blotting with antibodies, α-pTrkB or α-TrkB, against phosphorylated TrkB or TrkB, respectively. Note that the fusion receptors were phosphorylated only in the presence of BDNF. (B) CHO cells expressing GFP-TrkB were serum-starved for 3 h as described in Methods. Fluorescence signal of GFP were observed by confocal microscopy. This figure represents > 10 similar images observed. Bars, 10 μm. (C) CHO cells expressing GFP-TrkB were serum-starved, and then were incubated with culture media containing 0.45 M sucrose for 20 min at 37°C. After being incubated in the presence (+) or absence (-) of 100 ng/ml of BDNF for 5 min at 37°C, the cells were treated with (+) or without (-) the chemical cross-linker BS³ as described in Methods. The cells were lysed and subjected to Western blotting, with antibody against TrkB. This figure represents similar results of three independent experiments. Alpha-tubulin was visualized as a reference. The positions of molecular weight standards are shown at the right of each panel.