Figure 4

MUC16 decreases TRAIL-R2 expression and blocks caspase-8 activation at the DISC. (A) Monolayers of Ctrl scFv- and MUC16 scFv-expressing cells were detached using EDTA, washed with PBS and fixed with paraformadehyde 4% in PBS. Cells (non-permeabilized) were incubated with anti-TRAIL receptors R1, R2, R3 and R4 antibodies. Expression of TRAIL receptors was determined by flow cytometry and expressed as median fluorescence units. Results are from three independent experiments. (B) Lysates from Ctrl scFv- and MUC16 scFv-expressing OVCAR3 cells were immunoblotted with TRAIL-R2 antibody. (C) Lysates from empty vector (EV)- and MUC16CTD-expressing SKOV3 cells were immunoblotted with TRAIL-R2 antibody. (D) Ctrl scFv- and MUC16 scFv-expressing OVCAR3 cells were incubated with FLAG-tagged TRAIL (1 μg/ml) for various times and the DISC was immunoprecipitated with anti-FLAG antibody. The membrane was immunoblotted with anti-caspase-8 antibody or with anti-TRAIL R2. Data are representative of at least two independent experiments. (E) Empty vector (EV)- and MUC16CTD-expressing SKOV3 cells were incubated with FLAG-tagged TRAIL (2 μg/ml) and the DISC was immunoprecipitated with anti-FLAG antibody. The membrane was immunoblotted with anti-caspase-8 antibody or with anti-TRAIL R2. Data are representative of at least two independent experiments.