1% Coomassie brilliant blue R250 (CBB), 40% (v/v) methanol, and 1% glacial acetic acid solution, followed by destaining in 50% (v/v) methanol. Total proteins of the cells were solubilized for 2 h at 4 °C in a buffer containing 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 2.5% (w/v) sodium deoxycholate, 2% (v/v) NP-40, 1 mM N, N, N′, N′-ethylenediaminetetraacetic acid
(EDTA), protease inhibitors (1 mM PMSF, 1 μg mL−1 aprotinin, 1 μg mL−1 leupeptin, and 1 μg mL−1 pepstatin), and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF). The phosphoproteins were trapped on Phos-tag agarose phosphate-affinity beads and isolated according to the methods reported by GSI-IX in vitro Kinoshita-Kikuta et al. (2009). For LC-MS/MS analysis, the blots used in Phos-tag detection assays were incubated in 1 N aqueous NH3 for 15 min three times to remove the biotinylated Phos-tag complex (Nakanishi et al., 2007). Prior to LC-MS/MS analysis, the protein bands of interest on the blotting membranes were cut out and digested with 10−7 M lysyl endopeptidase (Wako) for 1 h at 37 °C. Peptides
produced by protease digestion were separated by a 0–40% linear acetonitrile gradient for 60 min, followed by analyses with a Waters UPLC Xevo Qtof. Obtained data were processed with ProteinLynx Global Server 2.4 and searched against Alveolata protein entries in the Uniprot Knowledgebase (UniprotKB) Selleck Adriamycin and Alveolata protein records in Entrez. When cells of C. cucullus cultured for 1–2 days were stimulated to encyst by
overpopulation in the presence of 0.1 mM Ca2+, the phosphorylation level of several proteins was enhanced within 1 h after onset of encystment induction (Fig. 1a, ‘P-tag’ right lane). In this case, the integrated optical density between in two lanes of CBB-stained blots (Fig. 1a, ‘CBB’) determined by NIH Image analysis was almost equivalent, indicating that an elevation of the Phos-tag signal (Fig. 1a, ‘P-tag’) should not be attributed to a difference in amounts of proteins between the lanes, but rather to a real enhancement of the protein phosphorylation level. In most of these cases, the increased phosphorylation Sclareol level was maintained for at least 12 h (data not shown). In the preparation of the protein samples shown in Fig. 1a, the sample buffer for SDS-PAGE did not contain inhibitors of proteases or phosphatases. Therefore, the phosphorylated proteins detected in this assay may have contained protein fragments digested by endogenous proteases, and some of the proteins may have been dephosphorylated by endogenous phosphatases during sample preparation. Therefore, as shown in Fig. 1b, the phosphorylation assay of the encystment-induced Colpoda proteins was re-examined in the presence of protease and phosphatase inhibitors (PPI).
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