Fig. 1. uPAR expression, uPA binding and membrane partners of cells expressing HD mutant uPAR. (A) FACS analysis of uPAR expression in H1299 cells. Control, uPAR-knockdown, WT and HD cells were harvested and incubated with antibodies against uPAR, followed by APC-conjugated secondary antibodies. (B) uPA 1-48-Fc binding to H1299 cells. uPAR-knockdown (shu), WT and HD cells were acid-washed to remove bound endogenous uPA, cells were incubated with 1-48-Fc (0-50 nM) at 4°C for 1 hour and the bound 1-48-Fc was detected by anti-Fc-APC. The mean fluorescence was plotted. Data represents mean ± s.d. from three independent experiments. (C) Co-immunoprecipitation of uPAR with caveolin, V integrin, or EGFR. H1299 cells expressing WT and HD uPAR were lysed in 1% Triton X-100 lysis buffer and the lysates were immunoprecipitated with mAb to uPAR (R2). The lysates and immunoprecipitates were then separated by SDS-PAGE and blotted for uPAR (399R), caveolin pAb, V integrin pAb or EGFR pAb. The experiment was performed at least three times with similar results.