Figure 4. Pro-cath-D and LRP1β partially co-localize at the cell surface in transfected COS cells and human mammary fibroblasts.

(A) Co-transfected pro-cath-D and LRP1β co-localize partially in punctuate structures. Non-permeabilized COS cells transiently co-expressing cath-D and Myc-LRP1β were stained with a monoclonal anti-cath-D recognizing only the proform (red) and a polyclonal anti-Myc tagged LRP1β (green) antibody. DNA was visualized by incubation with 0.5 μg/ml cell-permeant Hoechst 33342 dye (blue). Panels a shows LRP1β staining. Panel b shows pro-cath-D staining. Panel c shows the double-staining pattern. Arrows indicate pro-cath-D/LRP1β co-localization. Bars, 15 μm.

(B) Immunogold-labeled pro-cath-D and LRP1β co-localize at cell surface in COS transfected cells. COS cells transiently co-expressing cath-D and Myc-LRP1β were double stained with a monoclonal anti-pro-cath-D antibody recognizing only the proform and conjugated to 6 nm gold particles (thin arrows), and with a polyclonal anti-LRP1β antibody conjugated to 15-nm gold particles (thick arrows). Panels illustrate co-localization at the plasma membrane (PM). High magnifications of the boxed regions are displayed. Bars, 50 nm.

(C) Immunogold-labeled pro-cath-D and LRP1β co-localize at the cell surface and in vesicular-like structures in human mammary fibroblasts. COS cells were transfected with cath-D, and 48 h post-transfection conditioned medium containing 15 nM of pro-cath-D was added to HMF cells. HMFs incubated with pro-cath-D for 24 h were double stained with a monoclonal anti-pro-cath-D antibody recognizing only the proform and conjugated to 6-nm gold particles (thin arrows), and with a polyclonal anti-LRP1β antibody conjugated with 15-nm gold particles (thick arrows). Panels illustrate co-localization at the plasma membrane (PM). High magnifications of the boxed regions are displayed. Bars, 50 nm.