Immunoblotting in TNF-α-Stimulated NCI-H292 Cells

The cells (5×10⁴ cells/well) were incubated with AH (0, 20, 40, and 80 μg/ml) for 1 h, followed by incubation in the presence of TNF-α (30 ng/ml). After 1 h incubation, the cells were washed with PBS and protein was collected by using M-PER Extraction Reagents (Thermo Scientific) containing protease and phosphatase inhibitor cocktail. The expression levels of p-IκB-α, p-p65NF-κB and β-actin (loading control) were determined using immunoblotting as described above.