Cells were harvested at the end of the treatment, immediately frozen in liquid selleck chemical N2 and stored at 80 C until use. Medium alkalinization response assay To determine the UV B induced medium alkalinization, pH of the culture medium was measured from 0 to 120 min after 5 min of irradiation. UV B induced medium alkalinization response was calculated as the differ ence in pH between the untreated controls and the respec tive UV B irradiated samples as described. Measurement of H2O2 production H2O2 production was measured using cell permeable flu orescent probe 2, 7 dichlorodihydroflurescein diacetate by monitoring the increase in fluorescence by oxidation of DCFH to DCF as described by Pauw et al. The 2. 5 M DCFH DA was added to the cell suspension cultures immediately after UV B irradiation.

After UV B irradiation for different time periods, the increase in intracellular H2O2 levels was measured by monitoring the increase in fluorescence after 15 min with 488 nm excitation and 525 nm emission wavelengths in a luminescence spectrometer. To identify the events that inhibit the UV B induced H2O2 production, various inhibitors were added for 10 min prior to 5 min UV B radiation. Preparation of the cell extract Treated cell suspensions were collected by centrifugation, frozen separately in liquid nitrogen, and stored at 80 C until further use. Samples were thawed to 4 C and ultra sonicated in a buffer contain ing 50 mM HEPES KOH pH 7. 6, 2 mM DTT, 1 mM EDTA, 1 mM EGTA, 20 mM glycerophosphate, 20 % glycerol, 1 mM Na3VO4, 1 mM NaF and one tablet of complete pro tease inhibitors per 50 ml of buffer solution.

Homogenates were centrifuged at 12,000 rpm at 4 C for 25 min. The supernatant was used imme diately as a source of total soluble proteins to determine the activities of CDPK and MAPK. The total protein in the supernatant was estimated by the method of Bradford using BSA as a standard. Protein kinase assays Total soluble proteins extracted from C. roseus cells were assayed for CDPK and MBPK substrate phosphorylation activities according to the method of Putnam Evans et al. with slight modifications. Equal amounts of protein were taken and reactions were carried out in a total reac tion volume of 30 l kinase assay buffer for 30 min at room temperature. Sub strate phosphorylation assays were done by adding 50 g of myelin basic protein or histone IIIS, respectively, to the same reaction buffer as mentioned above.

The reaction was terminated by addition of electro phoresis sample loading buffer. After electrophoresis on 12 % SDS polyacrylamide gels, the phosphorylated MBP and HIIIS were visualized by autoradiography. CDPK and MBPK activities were determined by in gel Batimastat kinase assays using histone IIIS and myelin basic protein as substrates, respectively as described previously.