Fig. 6. Cleavage of the ErbB2 kinase domain is inversely correlated to internalization. (A) The upper left histogram shows r²_c values from cells treated with 3 µM geldanamycin for 4 hours and untreated cells. The lower histogram shows similar r²_c values for cells that in addition have been treated with the pancaspase inhibitor zVAD-fmk for 4 hours. The r²_c values were calculated for individual cells after staining as described in Fig. 1A. P-values from two-sided Mann-Whitney U-tests are shown. Note that zVAD-fmk inhibited ErbB2 cleavage. (B) The r²_i values from the same cells as in A, showing that zVAD-fmk has no effect on ErbB2 internalization. (C-E) Scatter diagrams, where each dot in the scatter diagram represents r²_i and r²_c values from a single SK-BR-3 cell treated as indicated and stained as described in Fig. 1A. (C) Untreated cells, cells treated with 3 µM geldanamycin for 1, 2 or 4 hours. (D) The cells were pre-treated with 50 µM zVAD-fmk and either added 3 µM geldanamycin or left in zVAD-fmk for 4 hours. The two images to the right show two different situations where either the internalization is dominant and no cleavage can be observed or where cleavage dominates and internalization is limited, which together with the scatter diagram suggest that these processes are competing events. (E) The cells were treated with 200 nM bafilomycin for 1 hour, then 3 µM geldanamycin was added for 4 hours. It is seen that cells with much intracellular ErbB2 had little cleavage and vice versa.