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Fig. 5. (A) Long-term PMA treatment leads to downregulation of PKC
and PKC
. KG1 and K562-ALCAM cells were cultured for 24 hours in presence or absence of PMA (50 nM) to induce downregulation of PKC. PKC expression was analyzed by western blotting. PKC isotypes and are both downregulated upon culturing in the presence of PMA in both cell lines. Expression of PKC isotypes 
and 
was not affected (data not shown). (B) PMA culturing inhibits cytoskeleton-dependent ALCAM-mediated adhesion. KG1 cells were cultured for 16 hours in the presence (white bars) or absence (black bars) of 50 nM PMA. Subsequently, induction of adhesion by pre-incubation of the cells for 30 minutes at 37°C with CytD (2.5 µg/ml) or LatA (5 µg/ml) was determined, in the presence or absence of the blocking antibody AZN-L50 (10 µg/ml). Downregulation of PKC upon culturing with PMA inhibits CytD- and LatA-induced adhesion to background levels. Similar observations were made for K562-ALCAM (not shown). (C) PMA culturing does not affect the overall ALCAM expression. ALCAM expression on KG1 cultured in the presence or absence of PMA (50 nM) was determined by flow cytometry. Shaded histograms represent isotype control staining and white histograms represent ALCAM staining with mAb AZN-L50.
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