Fig 4.
Association of stabilin-2 and its cytoplasmic deletion mutant with integrin β5. (A) 293FT cells were transiently transfected with stabilin-2–FLAG- and/or integrin β5-expressing vector. IB, immunoblotting. (B) Stabilin2ΔC-FLAG- and/or integrin β5-expressing vector was transfected into 293FT cells. The lysates were subjected to immunoprecipitation (IP) using either anti-integrin β5 (left) or anti-FLAG antibody (right). The immunoprecipitates were then analyzed by immunoblot analysis using anti-integrin β5 and anti-FLAG antibodies, respectively. Expression of stabilin-2 and integrin β5 proteins in total cell lysates was examined by immunoblot analysis (TCL, total cell lysate). A representative result of three independent experiments is shown. (C) Interaction between stabilin-2 and integrin β5 was assessed by FRET analysis. Three fluorescent images of COS7 cells expressing stabilin-2–seGFP and Venus-β5 were obtained, FRETC was calculated, and the results are displayed using quantitative pseudocolor (see Materials and Methods). In the lower panels, stabilin-2–seCFP and Venus alone were overexpressed in COS7 cells, and their interaction was assessed by FRET analysis as a control. (D) Colocalization of stabilin-2 (red) and endogenous integrin αvβ5 (yellow) in phagocytic cup area. Stab-2/L cells were allowed to ingest NBD-PS-coated beads (green). The cells were fixed and immunostained for Myc–stabilin-2 and αvβ5. (E) Colocalization of endogenous stabilin-2 (red) and integrin αvβ5 (green) in phagocytic cup area of stabilin-2-expressing native cells (human monocyte-derived macrophages). Stab-2/L cells were allowed to ingest NBD-PS-coated beads (blue). The endogenous stabilin-2 and αvβ5 were stained using the corresponding antibodies, as described in Materials and Methods. DIC, differential interference contrast.