Receptor chimeras with the transmembrane and stalk regions from TRAILR1 and TRAILR2 show comparable ligand-induced receptor aggregation and internalization.

<p><b>A.</b> MF-TM1, MF-TM2 and MF-TNFR1-Fas cells were transiently transfected with a construct expressing human FADD-eGFP in the presence of 20 µM z-VAD-fmk. The following day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods, fixed and examined by confocal laser-scanning microscopy. Shown are optical sections through the center of representative cells. <b>B.</b> Adherent cells were incubated with ¹²⁵I-TNF (30 ng/ml) for 1 h on ice, followed by incubation at 37°C and 5% CO₂ for the indicated times. Subsequently cells were washed with PBS, followed by washing with acidic buffer (pH = 3.0) to disrupt ligand/receptor interactions on the cell surface, or again with PBS (pH = 7.0). For non-specific binding (NSB) a 200-fold excess of unlabeled TNF was added during the first incubation step. Radioactivity of the cell lysates was then quantified in a γ-counter. Data points represent mean values ± SD from three independent experiments each performed in duplicates.