For immunoprecipitation, soluble proteins that had selleck chem been made up to a total vol ume of 100 l with immunoprecipitation buffer were incubated in a 1. 5 ml eppendorf tube with 5 g of mono clonal anti phosphotyrosine or anti phosphoserine anti body for 2 h at 4 C. For CDPK assay the same immunoprecipitation buffer was used without EDTA and EGTA. For reactions with competitor phosphoaminoac ids, antibodies were preincubated for 30 min at room temperature with 1 mM of the phosphoaminoacid. Approximately 25 l packed volume of recombinant pro tein A, immobilized on agarose, was added, and incuba tion continued for another 2 h at 4 C. The immunoprecipitated MBPK and CDPK were pelleted by centrifugation at 12,000 g for 10 min and washed two times with immunoprecipitation buffer.
The samples were boiled for 2 min and separated by electrophoresis on 10 % SDS gels with MBP or H IIIS, respectively and in gel kinase assays were done as described above. RNA isolation and RT PCR analysis Total RNA from cells of C. roseus was isolated using the Qiazol reagent following the manufacturers instructions. The RNA samples were quan tified by spectrophotometry at 260 and 280 nM and visual inspection in agarose gels. DNA was removed from total RNA sam ples by treatment with RNase free DNase I. Reverse tran scription was carried out in a 20 l reaction containing 1 g of total RNA, 5 g oligo d 16 18 primer, MuMLV reverse transcriptase, RNasin, 0. 5 mM dNTPs and MuMLV reverse transcriptase reaction buffer at 37 C for 1 h, and terminated by heat ing at 70 C for 10 min.
After the RT reaction, the cDNA was subjected to PCR reactions. One l of the RT reaction was used for PCR in 20 l containing 0. 4 U of Taq DNA polymerase, 0. 1 mM dNTP, 200 M of each dNTP and 100 pM of each primer in a 1�� reaction buffer. Reactions were amplified for a total of 15 cycles on the Minicycler using 94 C for denaturation, 55 C for annealing for Tdc and Str and 52 C for annealing for Rps9 and 72 C for extension, following a fur ther 5 min extension. The RT PCR products were sepa rated by electrophoresis on 1 % agarose gels, stained with ethidium bromide, and photographed under UV light using Alpha Imager 2200. RT PCR analysis of ribosomal protein 9 was used as control to check RNA integrity and accuracy of loading. The RT PCR products of the expected sizes 1. 5, 1. 2 and 0.
63 kb respectively was obtained for Tdc, Str and Rps9 and their identity confirmed by sequenc ing. Quantification of catharanthine by HPLC analysis The extraction of terpenoid indole alkaloids and quantifi cation of catharanthine using HPLC were according to Dacomitinib Schripseme and Verpoorte. The amount of catharan thine was finally reported as mg g 1 DW cells. Background Endothelin 1 is a potent vasoconstrictor produced by endothelial cells.