FIG 3 .

Anti-CK8 antibodies block Pet binding to plasma membrane of epithelial cells. (A) Anti-CK8 antibodies are more effective at blocking Pet binding than anti-CK10 antibodies. HEp-2 cells seeded in 96-well plates were washed, fixed (2.5% PFA), and blocked (3% BSA). Cells were incubated for 1 h with anti-CK10 or CK8 antibodies at different concentrations, and then cells were incubated with 100 nM of Pet per well at 37°C for 90 min and washed three times with PBS. The amount of membrane-bound Pet was determined by ELISA using anti-Pet antibodies followed by an anti-rabbit secondary antibody conjugated with HRP, with readings made at 490 nm. (B) Anti-CK8 antibodies inhibit Pet binding to plasma membrane. HT-29 cells were seeded, washed, and fixed as described for panel A. Cells were incubated with monoclonal anti-CK10 or anti-CK8 antibodies at a dilution of 1:10. Then, cells were incubated at 37°C for 90 min with different concentrations of Pet per well. Following 3 washes with PBS, the amount of membrane-bound Pet was determined by ELISA using rabbit anti-Pet antibodies followed by an anti-rabbit secondary antibody conjugated to HRP, with readings made at 490 nm. Values are the means of triplicate measurements from three independent experiments. (C) Pet interacts with recombinant CK8. ELISA plates coated with recombinant CK8 (20 nM) were incubated for 2 h with increasing concentrations of Pet and then washed with PBS to remove nonspecific binding. Finally, the amount of CK8-bound Pet was determined using rabbit anti-Pet antibodies followed by anti-rabbit secondary antibody conjugated to HRP, with the reading made at 490 nm. Wells without (w/o) CK8 were used as a negative control. Values are the means of triplicate measurements from three independent experiments.