To determine the time course of NFκB phosphorylation, first a titration was run on the concentration of the test compound; here we use TNF-α as an example. In a sterile, black 96-well plate, MDA-MB-231 cells were plated at a cell density of 20,000 cells per well and allowed to adhere overnight. The next day, the culture medium was removed and replaced with medium dosed with TNF-α at the following concentrations: 0.0E+0, 3.8E-4, 1.5E-3, 6.1E-3, 2.4E-2, 9.8E-2, 3.9E-1, 1.6E+0, 6.3E+0, 2.5E+1, 1.0E+2, and 4.0E+2 ng/mL. The dosed culture medium was prepared from a stock solution of 10,000 ng/mL TNF-α in dH2O. Triplicate wells were incubated for 20 minutes with the TNF-α. The pNFκB and total protein was then quantified using the NFκB Phosphorylation Assay Kit (ENFKB-100). The pNFκB values were normalized to the total protein content of the sample.