HUCL (A, B & D) and Primary (C) cells were incubated with various concentrations of SpA for 1 h (A), or 100 μg/ml for different time points (B & C) with unstimulated cells as control. A, B, and C, Western blotting of total cell lysate for phospho-IκB-α (IκB-α) and total IκB-α. D. NF-κB p65 DNA binding activity in HUCL nuclear extracts measured by ELISA using a Colorimetric Transfactor p65 kit.. Results shown are representative of three independent experiments.