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Fig. 4. JAM-A is internalized in neutrophils upon fMLP treatment. (A) Immunofluorescence internalization analysis of JAM-A endocytosis from the plasma membrane in fresh human neutrophils, either treated (b) or not (a) with fMLP. JAM-A internalized vesicles (red) are more abundant in activated cells. Each panel is a single projection of a z-stack of three confocal sections (stack z-spacing, 0.3 µm). Cell nuclei were counterstained with DAPI (blue). Scale bars: 5 µm. (B) Freshly purified neutrophils were labeled at 4°C with cleavable biotin and left untreated or stimulated with fMLP for 5 and 10 minutes at 37°C before surface biotin cleavage with GSH, and lysis. Biotinylated proteins were precipitated with streptavidin-agarose followed by western blot analysis with anti-JAM-A (BV22 mAb) and anti-
L integrin (clone 27) antibodies. (C) Densitometric analysis of internalized JAM-A and L integrin normalized for their total amount. The figure shows one representative experiment out of five performed.
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