FIG. 4.

Trk-deficient PC12-NNR5 (NNR5) cells transfected with TrkC, TrkB, and EV respond appropriately to corresponding ligands. TrkB and TrkC were cloned from human RNA into the bicistronic mammalian expression vector pIRES-dsRed, which was used to transfect PC12^NNR5 cells. (A) NNR5^TrkC, NNR5^TrkB, and NNR5^EV (EV) cells were sorted by FACS for dsRed expression to obtain similar expression levels; rectangles represent the cells selected from each population. The x and y axes represent channels that detect green fluorescent protein (GFP) and red fluorescent protein (RFP), respectively. FL, fluorescence. (B) P-Erk was examined by Western blotting with transfected cells cultured in serum-free medium overnight and treated with NT-3 or BDNF (100 ng/ml) for 12 min at 37°C. The upper and lower bands in each blot represent Erk1 (MAPK3) and Erk2 (MAPK1), respectively. (C) Transfected cells were cultured in medium with or without NT-3 (100 ng/ml) or BDNF (100 ng/ml) for 3 days, fixed, probed with anti-neurofilament primary antibody and Alexa 488 secondary antibody, and imaged (magnification, ×20) to visualize neurite extension. Experiments were repeated multiple times at various points to ensure that cells maintained correct responsiveness.