Figure 4 Effect of CCAD on AchE activity in neuronal-like PC12 cells. (A) PC12 cells were seeded into 24-well plates at 30,000 cells/well in maintenance medium and cultivated overnight. The next day cells were treated with NGF (50 ng/mL) and donepezil (10 μM), FGD or CCAD at 0.5 mg/mL. Cells were further cultivated for 6 days. At the end of cultivation, AchE activity of cells was measured by the fluorometric AchE assay as we described in the Materials and Methods section. (B) PC12 cells were treated as same as the description in Figure 3. At the end of cultivation, AchE activity of cells was measured by the fluorometric assay. (C) Membrane potential of CCAD + NGF-treated cells was tested by the patch clamp assay. Experiments were repeated 3–5 times independently. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by the least significant difference test). AchE: Acetylcholinesterase; CCAD: enzyme-digested Colla Corii Asini; Em: emission; Ex: excitation; FGD: enzyme-digested fish gelatin; NC: negative control; NGF: nerve growth factor.