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Fig. 1. (A) Flow-cytometric analysis of ALCAM expression by KG1 and K562-ALCAM cells. The shaded histograms represent the isotype control, whereas the white histograms represent ALCAM staining with AZN-L50. (B) ALCAM-mediated adhesion of both K562-ALCAM (white bars) and KG1 cells (black bars) to immobilized ALCAM-Fc requires disruption of the actin cytoskeleton. The cells were pre-incubated for 30 minutes at 37°C with or without CytD (2.5 µg/ml) or LatA (5 µg/ml) and subsequently allowed to adhere to an ALCAM-Fc coated plate (250 ng/ml ALCAM-Fc) for 45 minutes at 37°C in the presence or absence of the ALCAM-blocking mAb AZN-L50. Non-ALCAM antibodies did not affect binding, showing that the observed adhesion is ALCAM-mediated (data not shown). Specific adhesion is expressed as the mean percentage (±s.d.) of adherent cells from triplicate wells after subtraction of the adhesion in the presence of the blocking mAb AZN-L50. Data are representative of three experiments. (C) CytD treatment does not alter the affinity of K562-ALCAM (circles) and KG1 cells (triangles) for ALCAM-Fc. Cells were treated with (open symbols) or without (closed symbols) CytD (as in B) and subsequently incubated with the indicated dilutions of ALCAM-Fc. The percentage of cells that have bound ALCAM-Fc is detected by flow cytometry using a FITC-conjugated secondary goat-anti-human-Fc antibody.
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