Modulation of Smad, ERK, Akt and NF-κB activation by BMP-2. A and B. TOV-2223 cells were stimulated in complete media for indicated times with 50 ng/ml BMP-2 (left) or for 20 min with increasing doses of BMP-2 (10, 50 or 100 ng/ml). TOV-112D and TOV-1946 cells were stimulated for 20 min with 50 ng/ml BMP-2. Cytoplasmic (EC) or nuclear (EN) extracts were subjected to western-blotting using anti-phosphoserine Smad1/5/8 and anti-phospho-tyrosine Erk1/2 antibodies. *Cells were stimulated with 50 ng/ml BMP-2 and 0.5 ng/ml Noggin C. Total extracts were subjected to western-blotting using anti-phosphoserine 473 Akt. TOV-2223 cells were stimulated for 5 or 20 min with 50 ng/ml BMP-2. TOV-1946 cells were stimulated with 50 ng/ml BMP-2 for 20 min. D. Total extracts from TOV-2223 cells were immunoprecipitated with anti-p65 antibody and loaded on an 8% polyacrylamide gel. Western-blotting was performed with anti-phosphoserine 536 p65. E. Cells were cotransfected with Renilla and 3κB-conA-luc vectors. Eight hours after transfection, cells were incubated with fresh media in the presence or absence of 50 ng/ml BMP-2 with or without 0.5 ng/ml Noggin or 10 ng/ml TNFα for 24 hrs. Cells were assayed for luciferase activity. Relative firefly luciferase activity was the ratio of luciferase activity in treated cells to that of non-treated cells. All experiments were repeated three times with similar results.