FIGURE 4.
Plg-RKT is an integral plasma membrane cell surface protein. A, phase partitioning of Plg-RKT. Non-transfected PC12 cells were solubilized in 3% Triton X-114. After heating at 37 °C and separation of the phases by centrifugation, an aliquot of both phases was electrophoresed and Western blotted with polyclonal anti-Plg-RKT. An immunoreactive band corresponding to the Mrapp of Plg-RKT was detected in the detergent phase, but not in the aqueous phase. In controls for the method, when the cell lysates were spiked with BSA and subjected to phase partitioning, BSA was detected in the aqueous, but not the detergent, phase (data not shown). No bands were detected using control preimmune IgG (data not shown). B, co-immunoprecipitation (IP) of Plg-RKT with uPAR. Membrane fractions from non-transfected PC12 cells were prepared as described under “Experimental Procedures” and immunoprecipitated with polyclonal anti-Plg-RKT IgG or preimmune IgG. Membrane fractions and immunoprecipitates were electrophoresed on SDS-PAGE and immunoblotted for uPAR and for Plg-RKT. No bands were detected using control preimmune IgG isotype control for immunoblotting (data not shown). C, FACS analysis of Plg-RKT expression on intact PC12 cells. Non-transfected PC12 cells were analyzed by dual-color FACS analysis as described under “Experimental Procedures.” Viable cells were gated from non-viable cells, and histogram plots of viable cells are shown. Dotted tracings = anti-Plg-RKT mAb IgG. Black tracings = isotype control IgG. Dashed tracings = autofluorescence (Auto).