Figure 3.

Effects of ceramide, NO, and cGMP on TNF-α-induced TRADD recruitment to TNF-α-R1. (A) Time course of TRADD recruitment induced by TNF-α (50 ng/ml)/CHX (1 μg/ml) revealed by Western blotting of the precipitates with an anti-TRADD Ab. The gel shown is representative of four individual experiments: 0, 2, 5, and 10 indicate the time (min) of cell exposure to TNF-α before immunoprecipitation. Thirty micrograms of whole-cell homogenate was loaded in lane L as an internal control. Migration of the Ig heavy chain (HC) and TRADD is specified on the right. (B) Cells were treated with TNF-α (50 ng/ml)/CHX (1 μg/ml) (control) with or without SNAP (300 μM), 8 Br-cGMP (3 mM), ODQ (1 μM), D609 (25 μg/ml) and C₂ Cer (50 μM) in various combinations, as indicated in the key. The specific bands recognized by the anti-TRADD Ab were analyzed by densitometry, and values are expressed as a percentage of those observed in the TNF-α/CHX-treated control (n = 5).