Figure 1. Cell surface clustering of CD151 in response to mAB 1A5.

The cell surface distribution of CD151 was visualized by fluorescent microscopy in NIH3T3 cells after transfection with CD151-GFP and culture in the absence or presence of mAB 1A5 (2μg/ml) for 2hr. (A) CD151-GFP visualized in the absence (left panel) or presence of mAB 1A5 (right panels, 2μg/ml for 2hr). Surface bound antibody was detected using TRIC-conjugated anti-mouse IgG post fixation. Insets in A emphasizing general cell surface (i and ii) v.s. areas of cell-cell contact (i′ and iii) are magnified in (B). (C) Surface CD151 was detected in sequential Brij99 and RIPA extractions with avidin-HRP after biotinylation of surface proteins and immunoprecipitating the tetraspanin from HEp3 cells cultured in the presence of a control IgG or mAB 1A5 for 24hr. (D) Live cell imaging was used to evaluate changes in CD151 distribution in response to mAB 1A5 binding. A549 cells transfected with CD151-GFP (green) were imaged every 5 min for 2 hr with the addition of Alexa 546-conjugated mAB 1A5 (red) added at t=60 min and flushed from the system at t=80 min.