Assessment of IκB-α and NF-κB protein levels.

(A) Total cellular proteins from SMC cultures treated for 1 to 30 minutes with OSM (10 ng/mL) and TNF-α (10 ng/mL) were separated on a 4% to 12% Bis-tris gradient gel and transferred to a PVDF membrane. IκB-α was visualized by incubating the filters with a polyclonal antibody to IκB-α. (B) SMC cultures were stimulated with OSM in the presence or absence of U0126, NF-κB inhibitor peptide, or NF-κB control peptide. Cytoplasmic and nuclear NF-κB were isolated as described in “Materials and methods.” NF-κB was visualized by incubating the filters with a monoclonal antibody to NF-κB. Experiments were repeated twice.