Fig. 4.

Effect of chronic MnCl₂ on TH activity and phosphorylation levels in differentiated N27 cells. Differentiated N27 cells were incubated with 0.1 μM, 0.3 μM or 1 μM MnCl₂ for 24 h. For measurement of TH activity, cells were exposed to 2 mM NSD-1015 for 1 h prior to MnCl₂ treatment. Cells were lysed after treatment, and extracts were used to determine L-DOPA levels by HPLC. A, TH activity expressed as pg DOPA/μg protein; B, TH activity expressed as percentage of control. The data represent a mean + SEM of six to eight individual measurements. Asterisks (*p<0.05) indicate significant differences between MnCl₂-treated cells and control cells. C, Western blot of P-TH-Ser40. Cell extracts from control and 1 μM MnCl₂-treated N27 cells were prepared and separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. TH antibody and phospho-specific antibodies directed against P-TH-Ser40 were used for immunoblotting. To confirm equal protein loading in each lane, the membranes were reprobed with β-actin antibody. The immunoblots were visualized using Amersham’s ECL detection agents.