FIGURE 3.

Chronic ethanol does not increase the transcription and translation of TH, but enhances TH protein stability. A, cells were treated without (lane 1, control) or with 100 mm ethanol for 12 or 24 h (lanes 2 and 3). TH mRNA expression levels were analyzed by RT-PCR with actin mRNA levels as an internal control. The histogram depicts the mean percentage change in the ratio of TH to actin ± S.D. from three experiments. B, cells were treated without (lane 1, control, and lane 3) or with 100 mm ethanol (lanes 2 and 4) for 24 h, and 30 μg/ml CHX was added as indicated for the last 12 h of the 24-h treatment (lanes 3 and 4). TH protein levels were analyzed as described above. The histogram depicts the mean percentage change in the ratio of TH to actin ± S.D. from three experiments. **, p < 0.01, compared with control. C, cells were treated without (control) or with 100 mm ethanol for 24 h before a pulse-chase procedure as described under “Experimental Procedures.” Cells were labeled with 25 μCi of [³⁵S]Met/Cys pro-mix cell labeling mix for 3 h and then incubated in the normal medium for the indicated chase times, followed by immunoprecipitation with anti-TH antibody, separation on an SDS-PAGE gel, and autoradiography. The histogram depicts the mean percentage change in radioactive signals of ³⁵S-labeled TH protein at each chase time to those at initiation of chase (0 time) from six experiments. *, p < 0.05; **, p < 0.01, compared with control.