Figure 1.

Regulated secretion of neurotrophins from PC12 cells transiently transfected with neurotrophin expression plasmids. Three days after transient transfection with neurotrophin expression plasmids using Lipofectamine, PC12 cells were washed twice, preincubated for 45 min in physiological buffer, and subsequently stimulated for 30 min at 37°C with high potassium buffer (KCl), physiological buffer alone (control), or buffer containing the indicated neurotrophin at 100 ng/ml. In some experiments, new protein synthesis was blocked by adding cycloheximide (CHX) during the 45-min preincubation and the 30-min stimulation period. After stimulation, supernatants were removed, cleared by centrifugation, and subjected to neurotrophin-specific ELISA. PC12 cells were transiently transfected with pBJ-5-NT-3 (A), pBJ-5-BDNF (B), pBJ-5-NT-4/5 (C), or pBJ-5-NT-3 myc (D). Experiments were performed in triplicate; error bars indicate ±SD.