Figure 7. α6 integrin recycled to cell-cell lateral membrane locations.

Surface α6 integrin was labelled with J1B5 rat monoclonal antibody in DU145 cell monolayer and allowed to internalize for 40 minutes at 37°C. Uninternalized label at the cell membrane is blocked using Alexa 568 conjugated anti-rat secondary antibody. J1B5 antibody bound integrin protected inside the cells was allowed to recycle back and subsequently reacted with Alexa 488 conjugated anti-rat secondary antibody. (A) Total membrane and internalized intracellular α6 integrin in permeabilized cells shown as control (red). (B) Uninternalized α6 integrin (red) and recycled α6 integrin (green) at 0 min, 10 min and 40 minutes of recycling and 40 min recycling with primaquine (PQ, recycling inhibitor) are shown in merged images with DAPI (blue) stained nucleus (left panel). Middle panel shows only recycled α6 integrin label in gray. Right panel is magnified images of boxed sections marked for recycled integrin localized at cell-cell membrane locations (white arrows), uninternalized integrin at cell-cell locations (closed white triangles) and lamellipodia at cell front (open white triangles). Images acquired by deconvolution microscopy and single Z-plane is shown. Bars, 20μm (C) Bar graph showing average pixel intensity of recycled label plotted for different timepoints of recycling at room temperature (RT) (**p<0.01, 10 field of views in 3 independent experiments).