NF-B and Matrix-Dependent Regulation of Osteopontin Promoter Activity in Allylamine-Activated Vascular Smooth Muscle Cells
Figure 2
Activation of NF-κB signaling in allylamine cells. Panel (a) shows NF-κB binding activity in control and allylamine cells over the course of a synchronization and mitogenic stimulation cycle. Cells were seeded at equivalent densities on plastic tissue culture dishes and nuclear extracts collected at multiple time points as noted. EMSA were performed using end-labeled NF-κB consensus oligonucleotide as a probe. Arrows denote the major NF-κB binding complexes identified. Similar results were seen in 2 independent experiments. C# denotes individual complexes. Panel (b) shows IκBα and IκBβ protein levels in control and allylamine cells over the course of a synchronization and mitogenic stimulation cycle. Cells were seeded at equivalent densities and serum restricted for 72 hr to synchronize in G0, and then released into growth by the addition of 10% fetal bovine serum. Crude protein extracts were harvested at various times after addition of complete medium. Protein extracts were electrophoresed, electroblotted onto nitrocellulose, and probed for each IκB. Similar results were observed in 3 separate experiments. C: control; A: allylamine. Panel (c) shows serine phosphorylation levels of immunoprecipitated IKKα and IKKβ in control and allylamine cells. Immunoprecipitated IKKα/β was electrophoresed and transferred onto two nitrocellulose membranes and blocked overnight in 5% milk. One membrane was probed for IKKα/β, and the other for phosphoserine, followed by incubation with horseradish peroxidase- (HRP-) labeled secondary. Membranes were then incubated with chemiluminescent substrate and visualized using the KODAK Image Station. Densitometry was performed using Kodak 1 D Image Software. □: control, ■: allylamine.
