Okadaic acid

Hydroxyurea, 5-Fluorouracil, cantharidin and propidium iodide were purchased from Sigma-Aldrich; MS-275, KU-60019, MK-8776, LY2603618, LBH589 and VE-821 from Selleck Chemicals; okadaic acid was purchased from Enzo Life Sciences; Annexin-V-FITC-conjugated was from ImmunoTools. Fmoc-protected amino acids and coupling reagents (HBTU, HOBt) were purchased from Novabiochem, Iris Biotech GmbH and Orpegen Pharma GmbH, respectively. Reagents for synthesis and solvents for chromatography (analytical grade) were from VWR International GmbH.

Purified human serum IgA (>20 mg/ml) was obtained from Cappel (Malvern, PA). It contained no detectable trace of IgG, IgM, or non-immunoglobulin serum proteins. Recombinant mouse IL-3 was produced in COS cells (25 (link)). Ultra-pure fibrinogen (Fib3) and human alpha thrombin was purchased from Enzyme Research Laboratories (Swansea, UK). Pharmacological inhibitors LY294002, SB216763 were purchased from BioMol (Plymouth Meeting, PA). PKB inhibitor 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate was obtained from Calbiochem (San Diego, CA), the GSK-3α/β inhibitor CHIR-99021 from Selleckchem and okadaic acid from Enzo Life Sciences. The PKCζ pseudo-substrate was purchased from Biosource (Camarillo, CA) or Santa Cruz biotechnology. Recombinant human GM-CSF (rhGM-CSF) was from Immunotools. PI-3 kinase construct p110-K227E was a kind gift of Dr. J. Downward (ICRF, London, UK). G418 and hygromycin B were purchased from Boehringer Mannheim (Germany). Incubation buffer contained 20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO₄, 1.2 mM KH₂PO₄, supplemented with 5 mM glucose, 1 mM CaCl₂, and 0.5% (w/v) HSA. All other materials were reagent grade.

Acrylamide (99%; for molecular biology), alternariol (96%; from Alternaria sp.), and doxorubicin hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA), while okadaic acid was acquired from Enzo Biochem Inc. (New York, NY, USA). For cell culture experiments, RPMI (Roswell Park Memorial Institute, Buffalo, NY, USA) 1640 medium, heat-inactivated fetal bovine serum, and penicillin/streptomycin (P/S) solution were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). For the cell viability assay, the CellTiter-Blue^® Cell Viability reagent was purchased from Promega Corporation (Fitchburg, MA, USA). The 4′,6-diamidine-2′-phenylindoledihydrochloride (DAPI), Alexa Fluor ™ 633 F(ab’)2 fragment of goat anti-mouse IgG (H+L), bovine serum albumin (BSA; Standard Grade Powder, Fraction V), and S9 fraction (20 mg/mL from liver of Sprague Dawley rats), which were used for the γH2AX assay, were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Anti-phospho-Histone H2A.X (Ser139) Antibody (clone JBW301) and glucose-6-phosphate di-sodium salt were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), while nicotinamide adenine dinucleotide phosphate (NADP) was purchased from Merck KGaA (Darmstadt, Germany).

MC-LR and MC-RR certified standards, the anti-adda antibody (AD4G2), and the assay kit for detection of MCs/NOD in water by PP2A were purchased from Abraxis, Inc (Warminster, PA, USA). MC-LF, NOD, cantharidin, okadaic acid, and calyculin A standard were purchased from Enzo (Farmingdale, NY, USA). Norcantharidin and phosphate buffered saline, pH 7.4 with Tween 20 (PBS-T) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All MCs and other PP2A inhibitors were stored at −20 °C. The antibody was stored at 4 °C. Acetonitrile was from Pharmaco (Dawsonville, GA, USA). Methanol and formic acid were obtained from Fisher Scientific (Waltham, MA, USA). Life Technologies Corporation (Grand Island, NY, USA) supplied the Dynabeads MyOne Streptavidin T1, Zebaspin 7K molecular weight cut off, 0.5 mL desalting columns, and EZ-link NHS-PEG4-biotin. The Thermomixer C, Protein LoBind 2 mL microcentrifuge tubes, Protein LoBind 1 mL deep well plates, and TwinTec Lo-Bind PCR plates were purchased from Eppendorf (Hauppage, NY, USA). V&P Scientific (San Diego, CA, USA) supplied the 96-well plate magnet (part number VP771HH-MC). The microcentrifuge tube magnet was purchased from Invitrogen (Waltham, MA, USA).

F12 and F12 (Coon’s modification) medium, latrunculin B, anti-α-tubulin mouse monoclonal antibodies (DM1A, Cat. no. T9026, lot no. 052M4837) and HRP-conjugated secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Lactacystin and okadaic acid were purchased from Enzo (Farmingdale, NY). Phosphoseek™ Phosphoprotein Enrichment kits were purchased from BioVision (Milipitas, CA). Mouse monoclonal antibodies against the V5 epitope tag (clone: SV5-Pk1, cat. no. MCA1360GA) were from AbD Serotec (Raleigh, NC) and rabbit monoclonal antibodies against WDR1 (Cat. No. EPR8793) were from AbCam (San Francisco, CA) or Santa Cruz (Dallas, TX) (Cat. No. B10). Mouse monoclonal antibodies against phospho-threonine (Q7, Cat. no. 1018223) and phospho-serine (Q5, Cat. no. 1018234) residues were purchased from Qiagen (Valencia, CA). Fetal bovine serum (FBS) was from Gemini Bio-Products (Woodland, CA). Polyclonal rabbit antibodies against rat serum albumin were generously provided by Dr. Ann Hubbard (Johns Hopkins University School of Medicine, Baltimore, MD).