Fig. 4. Adenovirally transduced nnr5 cells. (A) Time course of BDNF secretion from nnr5 cells double transduced with AdCMV-Re794Y and AdCMV-BDNF. Perfusion was performed as described in Figure 3. Two subsequent stimulations by 50 mM KCl and 100 ng/ml NGF were separated by a recovery period of 30 min. Fractions were collected every 5 min and the BDNF concentrations were determined by ELISA. The values presented are the mean ± SE (n = 4). (B) Expression of BDNF and Re794Y mutant in nnr5 cells co-transduced with AdCMV-BDNF and AdCMV-Re794Y. The expression of both BDNF and Re794Y was detected by probing with the hybridoma supernatant (9E10), which recognizes the myc epitope added to the C-terminal domain of BDNF, and with the anti-pan-Trk antibody, which recognizes the C-terminus of Re794Y. Cells were analysed by confocal microscopy. The pictures represent a single confocal scan along a given x–y plane. The BDNFmyc-infected nnr5 cells showed a discontinuous staining pattern characteristic for the localization of BDNF in ER and Golgi compartments (green). The staining obtained with an anti-pan-Trk antibody was localized predominantly at the plasma membrane (red).