Figure 3

Activation of Myelin Basic Protein Kinase (MBPK) activity by UV-B irradiation in C. roseus suspension cultured cells. Six-day-old cell suspension cultures were irradiated for 5 min with UV-B light (+) or left un-irradiated (-) as a control. Cells were harvested at the indicated time periods, crude extracts were prepared, and MBPK activity in the cell extracts was assayed using MBP as a substrate as described in materials and methods. (a) MBPK activity was carried out with an in vitro phosphorylation assay. The reaction mixtures were resolved by SDS 10% (w/v) polyacrylamide gel electrophoresis and the phosphorylated MBP was visualized by autoradiography. (b) MBPK activity in the cell extracts was determined by in-gel kinase assay with MBP as a substrate. Autoradiogram represents in-gel phosphorylation of MBP. (c) Detection of MBPK activity in immunoprecipitates from cell extracts using the anti-phosphotyrosine antibody. Lane 1 and 2 represent cell extracts subjected to in-gel kinase assay directly without immunoprecipitation. Lane 3 to 10 indicate the cell extracts subjected to immunoprecipitation with a monoclonal antibody specific for phosphotyrosine and the MBPK activity of the immunoprecipitates assayed by in-gel kinase assay. The phosphorylated MBP was visualized by autoradiography. Phosphotyrosine and phosphothreonine were used as competitor substrates to demonstrate the specificity of the antibody. Symbols (-) and (+) represent, untreated and treated of the indicated treatment.