NFκB Electrophoretic Mobility Shift Assay (EMSA)

Cells were plated at a density of 500,000 cells per well of a six-well plate and treated with media containing DMSO or dox/TMP for 48 hours before stimulation with IL-1α (25 ng/mL) for an additional 24 hours (in the presence of DMSO or dox/TMP). Nuclear fractionation and lysis were accomplished as described previously.³⁵ Nuclear lysates (5 μg) were incubated with 0.5 to 1.0 μL IRDye 700 NFκB probe (LI-COR) in binding buffer (10 mM Tris pH 7.5, 50 mM KCl, 3.5 mM DTT, 0.25% Tween 20, poly[dI*dC]) for 30 minutes at RT. Samples were then run on a nondenaturing 4% polyacrylamide gel in 0.5x Tris/Borate/EDTA buffer until resolved. The resulting gel was visualized on a LI-COR Odyssey CLx.