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Figure 3


Fig. 3. Loss of uPAR–β1-integrin association and change of cell morphology by H249A-D262A mutations on uPAR. (A) H, D or HD mutations in uPAR disrupt the formation of uPAR–β1-integrin complexes. H1299 cells expressing WT, H, D or HD uPAR were lysed in 1% Triton X-100 lysis buffer and the lysates were immunoprecipitated with monoclonal antibody to {alpha}3 integrin (P1B5) or {alpha}5 integrin (P1D6). The lysates and immunoprecipitates were then separated by SDS-PAGE and blotted for uPAR (R2) and integrin {alpha}3 or integrin {alpha}5. (B) uPAR co-clusters with {alpha}3 integrin but not {alpha}6 integrin. H1299 cells expressing WT or HD uPAR were plated on fibronectin and stained with mouse mAb to integrin {alpha}3 (P1B5) or rat mAb to integrin {alpha}6 (GoH3) on ice. The receptors were clustered with fluorophore-conjugated secondary antibody (Alexa Fluor 350 for {alpha}3 integrin or Alex Fluor 568 for {alpha}6 integrin) at 37°C or left nonclustered as controls. Cells were then fixed and stained with conjugated anti-uPAR antibody (Rhodamine-conjugated ATN615 for {alpha}3-integrin-clustered cells or ATN615 with Alexa Fluor 350 for {alpha}6-integrin-clustered cells). To facilitate visualization, images were converted to pseudo color using Zeiss LSM Image Browser software: integrin (green), uPAR (red) and sites of colocalization (yellow). The indicated areas in the merged images were magnified and shown on the right. The above experiments were performed three times with similar results. (C) Quantification of {alpha}3 integrin co-clustering with uPAR. Percentage colocalization (colocalized {alpha}3÷total {alpha}3x100) from three independent experiments was measured using Zeiss LSM Image Browser software: WT (n=21) and HD (n=24), Student's t-test, *P<0.005. (D) Phase images of H1299 cells expressing WT or HD uPAR. WT cells show robust lamellipodia formation that is absent in HD cells. The indicated areas were magnified and shown on the right. (E) Phase images of H1299 cells expressing WT uPAR with or without treatment of uPAR–β1-integrin blocking peptide β1P1 or its scrambled control. β1P1-treated WT cells show no lamellipodia formation.