Luciferase assay
Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega);
after protein concentration determination (Bradford assay) luciferase
quantification was assessed in luciferase assay buffer (470 μM
luciferin, 20mM Tricine, 0.1 Mm EDTA, 1.07 Mm
(MgCO3)4·Mg(OH)2×5H2O;
2.67 mM MgSO4×7H2O in
H2O, pH 7.8, with 33.3 mM DTT and 530 μM ATP), by
measuring luminescence emission with a Veritas luminometer (Promega).
The relative luminescence units (RLU), determined during a measurement
of 10 s time and expressed as luciferase units/μg protein, were
calculated as compared with blank control samples.