PLoS Med. 2006 October; 3(10): e420.
Luciferase AssayH838 cells stably expressing NQO1-ARE luciferase were seeded onto a 24-well dish at a density of 0.2 × 106 cells/ml for 12 h before transfection. WT-KEAP1 cDNA constructs along with the mutant cDNA constructs (G333C and L413R) were transfected into the cells along with pRL-TK plasmid expressing Renilla luciferase as a transfection control. Twenty-four hours after transfection, cells were lysed and both firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega).
J. Biol. Chem., Vol. 282, Issue 52, 37429-37435
Luciferase Assay—Dual luciferase assay was performed in triplicate according to the instructions of the manufacturer (Promega). The pGL2-p21A luciferase reporter under the control of the two p53-responsive elements in the p21 promoter was used (29). Briefly, 100 ng of pGL2-p21A luciferase reporter, 100 ng of pcDNA3 or pcDNA3-HA-p53, and 5 ng of Renilla luciferase assay vector pRL-CMV (Promega) were co-transfected into H1299 or MCF7 cells. The -fold increase in relative luciferase activity is a product of the luciferase activity induced by p53 divided by that induced by an empty pcDNA3 vector.
Plant Cell. 2004 May; 16(5): 1143–1162
Enzyme AssaysDHAR activity was assayed essentially as described (Hossain and Asada, 1984). Tobacco leaves were ground in extraction buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, and 1 mM MgCl2), and soluble protein was obtained after a 5-min centrifugation at 13,000 rpm. DHAR was assayed from an equal amount of protein as described (Bradford, 1976) in 50 mM K2HPO4/KH2PO4, pH 6.5, 0.5 mM DHA, and 1 mM GSH and its activity followed by an increase in absorbance at 265 nm. MDHAR (10−8 mol NAPDH oxidized/min/mg protein), SOD (units to inhibit nitroblue tetrazolium photoreduction by 50%), CAT (10−6 mol H2O2 reduced/min/mg protein), GR (10−8 mol NAPDH oxidized/min/mg protein), and APX (10−8 mol Asc oxidized/min/mg protein) activities were determined as described (Gainnopolitis and Pies, 1977; Aebi, 1984; de Pinto et al., 2000). Ascorbate oxidase activity present in apoplastic wash fluid (obtained as described below) was determined from the decrease in A265 (extinction coefficient of 14 mM−1cm−1) at 25°C in a reaction mixture containing 0.1 M sodium phosphate, pH 5.6, 0.5 mM EDTA, and 100 μM Asc as described (Pignocchi et al., 2003).
EMBO J. 2008 January 23; 27(2): 373–383.
Luciferase assay NF-κB activation was determined using 0.5 × 105 HEK293T cells transfected with expression plasmids in the presence of reporter plasmids, NF-κB-dependent pBxIV-luc and control pEF1BOS-β-gal, as described (Inohara et al, 2000; Kobayashi et al, 2002).
Mol Cell Biol. 2005 April; 25(7): 2808–2818.
Fluorometric assay of caspase activity. Caspase activity was determined as described previously (11). Briefly, cell lysates were incubated with 50 μM fluorogenic caspase-3 substrate DEVD-AMC or the caspase-8 substrate IETD-AMC in 200 μl of buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 10% sucrose, 0.1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, and 10 mM dithiothreitol. The release of aminomethylcoumarin was measured by fluorometry using an excitation wavelength of 360 nm and an emission wavelength of 475 nm.
