Fig. 4

TRAIL-induced receptor signaling complex formation and caspase-8 activation is equally effective under iso- and hypertonic conditions. a TRAIL-R1/-R2 signaling complexes were induced in HCT116 cells by stimulation with Fc-FLAG-scTRAIL (1 μg/ml) for the indicated periods of time in the presence and absence of NaCl (75 mM). Proteins associated with Fc-FLAG-scTRAIL were immunoprecipitated using protein G agarose and were analyzed together with the corresponding lysates by western blotting for the presence of the indicated proteins. Data shown are representative of two experiments performed. b and c HCT116 cells were treated with KillerTRAIL (32 ng/ml) in the presence and absence of NaCl (75 mM) for the indicated periods of time. Caspase-8 activity was assessed using the fluorogenic substrate (IETD)₂-R110. Please note that different concentrations of recombinant TRAIL required for caspase activity assays and co-immunoprecipitation experiments could affect the kinetics of caspase-8 activation. Shown are data points and mean ± SEM from three independent experiments. d HCT116 and SK-Mel-3 cells were challenged with KillerTRAIL (32 ng/ml) for 9 h in the presence and absence of NaCl (75 mM). After washing and lysis, western blot analyses were performed with antibodies specific for the indicated proteins. Detection of tubulin served as a loading control. RFU, relative fluorescence units