ATP was quantified by using a luciferine/luciferase-based measurement kit (Molecular Probes). In brief, a standard reaction solution was prepared with 400 µl 20 × reaction buffer, 80 µl 0.1 M DTT, 400 µl of 10 mM d -luciferine, 2 µl of 5 mg/ml firefly luciferase, and water to a final volume of 8 ml. Samples of RIPA homogenates (3.7 µl) were mixed with 71.25 µl of standard reaction solution, and the mixtures were then transferred to a 96-well microplate for measurement of ATP bioluminescence using a Varioskan LUX microplate luminometer (Thermo Scientific). ATP concentrations were calculated from a standard curve obtained with known amounts of ATP in standard reaction solution.
Varioskan lux microplate luminometer
Manufactured by Thermo Fisher Scientific
The Varioskan LUX microplate luminometer is a versatile instrument designed for luminescence-based measurements. It provides accurate and reliable detection of various luminescent signals, including bioluminescence, chemiluminescence, and fluorescence. The Varioskan LUX is capable of measuring multiple parameters simultaneously across microplates, enabling efficient high-throughput analysis.
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2 protocols using varioskan lux microplate luminometer
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Quantifying ATP via Luciferase Assay
2
Transient Transcriptional Activity Analysis
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For the transient transcriptional activity analysis, the full-length coding sequences of IbNAC3, NAP, or ANAC072 were inserted into the pGreenII 62-SK vector as an effector, including a CaMV35S-Rluc and internal reference REN. The promoter sequences of AtCHX25, AtRH33, MREL57, and ERA1 (same as in the Y1H experiment) were ligated into the pGreenII 0800-LUC double-reporter vectors (Hellens et al., 2005) . The recombinational construct was transformed into Agrobacterium strains (GV3101) and the cultured strains were resuspended into the medium (10 mM MES, 10 mM MgCl 2 , and 150 μM acetosyringone). Co-transforming the Agrobacterium strains containing effector and reporter vectors into N. benthamiana leaves and expressed for 2-3 days, then the luciferase activities of LUC and REN were detected by the Dual-Luciferase Reporter Assay System (Promega) based on the manufacturer's instructions using the VARIOSKAN LUX Microplate Luminometer (Thermo Fisher Scientific). Each promoter sequence in reporter vector with empty pGreenII 62-SK effector vector was set as a control, and the relative LUC/REN ratio was applied to represent the promoter activity. At least nine independent biological replications were carried out, related primers used were listed in Supplemental Table S6.
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