MC3T3-E1 cells express functional Fn14. (a) Cell surface expression of Fn14 on MC3T3-E1 cells. MC3T3-E1 cells were stained with anti-Fn14 monoclonal antibody (open histogram) or control Ig (filled histogram) followed by phycoerythrin-labeled rabbit anti-mouse Ig antibody and analyzed by flow cytometry. (b) TWEAK phosphorylates the NF-κB p65 subunit in MC3T3-E1 cells. MC3T3-E1 cells were stimulated with 100 ng/ml TWEAK or 10 ng/ml TNF-α (as a positive control) in the presence or absence of 1 μg/ml mouse Fn14-Fc chimera (Fn14-Fc) or control mouse IgG for the indicated time periods. The cell lysates were then subjected to immunoblotting with antibody specific for the Ser536-phosphorylated NF-κB p65 subunit. Fn14, fibroblast growth factor-inducible 14; Ig, immunoglobulin; NF-κB, nuclear factor-κB; TNF-α, tumour necrosis factor-α; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.