Cloning, characterization and immunolocalization of two subunits of calcineurin from pearl oyster (Pinctada fucata)

Calcineurin mediates the immune response of hemocytes through NF-κB signaling pathway in pearl oyster (Pinctada fucata)

“…As described in our former reports [10], CN (PP2B) enzyme activity in the hemocytes of P. fucata was measured using a nonradioactive serine/threonine Phosphatase Assay System (Promega), which determines the amount of free phosphate generated in a reaction by measuring the absorbance of a molybdate:malachite green:phosphate complex. The chemically synthesized phosphopeptide, RRA(pT)VA, was used as the substrate.…”

“…The samples for analyzing the levels of CNA in the various tissues (mantle, gonad, adductor muscle, gill, foot, and viscera) were prepared as described in the section of CN activity measurement in our former report [10]. The samples for detecting other items in the cultured hemocytes were prepared as follows: after the media from each culture dish were collected for analyzing the levels of IL-2 and NO, the hemocyte monolayer was lysed with 0.3 ml of ice-cold TNE lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1.0 mmol/L PMSF, 5 mg/L leupeptin, 5 mg/L aprotinin, 2 mg/L pepstatin) and agitated for 20 min at 4 C. Once the lysed hemocytes were centrifuged for 60 min at 10,000 g, 100 ml of the supernatants were collected for further centrifugation for 1 h at 100,000 g for CN activity assay, while the rest of the supernatants were collected for detecting levels of CA, and IkBa via Western blot, and the determination of iNOS with the Assay kit.…”

“…The rabbit polyclonal antibodies of CNA from P. fucata were prepared and purified as described previously [10]. Briefly, antibody against purified recombinant oyster CNA was raised by injection of 0.25 mg proteins in complete Freund's adjuvant into the rabbits, and then followed by two equivalent injections at a threeweek interval.…”

“…It is also demonstrated that CN mediates the control of gene expression through modulating the NF-kB signaling axis [8]. In molluscs, the sequence of CaM [9] and two subunits of CN [10] from Pinctada fucata have been cloned, while there is no genetic Abbreviations: Pf-CNA and Pf-CNB, two subunits of calcineurin in P. fucata; CaM, calmodulin; CsA, cyclosporin A; LPS, lipopolysaccharide; IL, interleukin; IL-2R, IL-2 receptor; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IkB, inhibitor of NF-kB; NF-kB, nuclear factor kappa B; IKK, inhibitor of NF-kB kinase; NFAT, Nuclear factor of activated T -cells; iNOS, the inducible isoform of nitric oxide synthase; NO, nitric oxide; DAB, 3,3 0 -diaminobenzidine; FITC, fluorescein isothiocyanate; CFU, colony forming units. information about NFAT in molluscs.…”

Cloning and characterization of two subunits of calcineurin cDNA in naked carp (Gymnocypris przewalskii) from Lake Qinghai, China

“…Protein yields were measured by BCA assay kit (Pierce, Rockford, IL, USA). Purified polyclonal antibody against the purified recombinant proteins was obtained following the reported method [17][18][19]. The purified antibody was used for Western blotting analysis and immunohistochemistry.…”