Fig. 5.

Cardiac glycosides have no effect on the binding of TNF-α to TNFR1. HeLa cells were treated with 100 nM oleandrin (I), 100 nM digitoxin (II), or 2,000 nM inactive cardiac glycoside (VIII) for 6 h. The cells were then collected for staining with biotinylated TNF-α and avidin-FITC. Soybean trypsin inhibitor that had been biotinylated to the same degree was used as a negative control. Neutralized TNF-α-biotin with anti-TNF-α blocking antibody was used as a specificity control. Receptor binding activity was determined by flow cytometric analysis with 488-nm wavelength laser excitation.