Fig. 4.

Overexpression of TrkA/TrkB fusion receptors in COS7 cells. a Phase contrast and fluorescence images of COS7 cells overexpressing TrkA/TrkB chimeric receptors, taken 2 days after transfection. The presence of GFP-positive cells in the culture (GFP) indicates that overexpression of TrkA/TrkB was successful. As a control, transfection of pBudCE4/GFP vector (Fig. 3b) was done. Scale bar, 100 μm (b, c) Expression and NGF-dependent activation of TrkA/TrkB fusion receptors. In b, 2 days after transfection, COS7 cells were cultured neurobasal medium containing 2% B27 supplement and 0.5 mM l-glutamine for 12 h and then exposed to NGF (100 ng/ml) for 5 min. The lysates (10 μg) collected from the COS7 cells expressing the indicated construct were subjected to electrophoresis on SDS-PAGE and immuno-blotting analysis was done with anti-phospho-Trk antibody recognizing the phosphorylated form of the tyrosine residue at the Shc binding site. Note that A/B-Wild and A/B-PLC-γ (−), but not A/B-Shc (−) and A/B-KN, were activated by NGF exposure. In c, cell lysates (10 μg) were further analyzed by immuno-blotting with pan-Trk antibody. The arrow in b and c indicates the band corresponding to phospho- and total TrkA/TrkB chimeric receptors, respectively