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. 2005 Jun 27;102(27):9631–9636. doi: 10.1073/pnas.0504097102

Fig. 4.

Fig. 4.

Cardiac glycosides suppress TNF-α-dependent recruitment of TRADD to TNFR1. (a) Reporter plasmids pNF-κB-luc and pRL-CMV were cotransfected into HeLa cells with effector plasmids HA-TRADD and Flag-TNFR1 overnight, then treated with 100 nM oleandrin (I) for 10 h. Cell extracts were then tested with the dual-luciferase reporter assay system. (b) HeLa cells were pretreated with 100 nM oleandrin (I), 100 nM digitoxin (II), or 2,000 nM inactive cardiac glycoside (VIII) for 6 h, then treated with 30 ng/ml TNF-α for 5 min. Cell extracts were used for immunoprecipitation (IP) with anti-TNFR1 and input control. (c) The plasmids HA-TRADD and Flag-TNFR1 were cotransfected into HEK293T cells overnight and then treated with the cardiac glycosides (CGs) 100 nM oleandrin (I), 100 nM digitoxin (II), or 2,000 nM inactive cardiac glycoside (VIII) for 10 h. Cell extracts were used for immunoprecipitation experiments with anti-Flag antibody and the input control. WB, Western blot.