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Fig. 7. Geldanamycin-induced ErbB2 internalization requires proteasomal activity. (A) Western blot of ErbB2 from SK-BR-3 cells treated with 3 µM geldanamycin as indicated. In the lower panel the cells were pre-treated with the proteasome inhibitor lactacystin (10 µM) for 1 hour and added geldanamycin. Note that ErbB2 is degraded after geldanamycin treatment, and that this depends on proteasomal activity. (B) Confocal images of SK-BR-3 cells treated with the proteasomal inhibitor lactacystin (10 µM) for 1 hour and then 3 µM geldanamycin (upper right image) for 4 hours. Strikingly, the internalization was inhibited compared to cells that were only treated with geldanamycin (upper left image). Likewise, if cells were co-treated with both bafilomycin (200 nM) and lactacystin (lower right image) before addition of geldanamycin there was no internalization of ErbB2, confirming the importance of proteasome activity for internalization and as a consequence lysosomal degradation. (C) Upper histogram shows r2i values from cells treated with 3 µM geldanamycin for 4 hours and cells that were treated with lactacystin for 1 hour and then geldanamycin for 4 hours. Lower histogram shows similar r2i values for cells that furthermore have been pre-treated with bafilomycin. The r2i values were calculated for individual cells after staining as described in Fig. 1A, and P-values are from two-tailed Mann-Whitney U-tests. (D) Confocal images showing cells treated with monensin and geldanamycin for 2 hours (left image). When cells were pretreated with 10 µM lactacystin before 10 µM monensin and 3 µM geldanamycin were added, the geldanamycin-induced accumulation of intracellular ErbB2 in cells with inhibited recycling (left image) was efficiently inhibited (right image), suggesting that proteasomal activity is needed for ErbB2 internalization. Bars, 20 µm.
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