Figure 4

Activation of CDPK in C. roseus suspension cultured cells in response to UV-B irradiation. 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 the activity of CDPK in the cell extracts was assayed using histone IIIS as a substrate as described in materials and methods. (a) CDPK was assayed with an in vitro phosphorylation assay. The reaction mixtures were resolved by SDS 10% (w/v) polyacrylamide gel electrophoresis and subjected to autoradiography. (b) CDPK activity in the cell extracts were determined by in-gel kinase assay with histone IIIS as substrate in the presence and absence of calcium. Autoradiogram represents in-gel phosphorylation of histone IIIS. Arrows show the molecular masses of two detected CDPK bands (c) Detection of CDPK activity in immunoprecipitates from cell extracts using anti-phosphoserine antibody. Lane 1 and 2 represent cell extracts subjected to in-gel kinase assay directly without immunoprecipitation. Lane 3 to 7 indicate the cell extracts subjected to immunoprecipitation with a monoclonal antibody specific for phosphoserine and the CDPK activity of the immunoprecipitates assayed by in-gel kinase assay. The phosphorylated histone IIIS was visualized by autoradiography. Symbols (-) and (+) represent, untreated and treated of the indicated treatment. Arrows show the molecular masses of two detected CDPK bands.