A Cell-Based Assay for IκBα Stabilization Using A Two-Color Dual Luciferase-Based Sensor

Abstract: A cell-sensor assay for stabilization of IκBα was developed in the activated B cell-like diffuse large B-cell lymphoma cell line OCI-Ly3. This cell line expresses known nuclear factor κB (NFκB) target genes due to high constitutive activity of IκB kinase (IKK), which phosphorylates the protein IκBα leading to proteasomal degradation of IκBα and activation of NFκB. The cell-sensor assay uses green and red light-emitting beetle luciferases, with the green luciferase fused to IκBα (IκBα-CBG68) and the red luciferase (CBR) present in its native state. The IκBα-CBG68 reporter functions as a sensor of IKK and proteasome activity, while CBR serves to normalize for cell number and nonspecific effects. Both reporter constructs were stably integrated and placed under the control of an inducible promoter system, which increased fold responsiveness to inhibitors when assay incubations were performed simultaneous to reporter induction by doxycycline. The assay was miniaturized to a 1,536-well plate format an...