Fig. 2. ErbB2 is internalized and degraded in lysosomes in response to geldanamycin treatment. (A,B) Confocal images of SK-BR-3 control cells and cells treated with 3 µM geldanamycin for 2 hours at 37°C. (A) Thirty minutes before fixation Alexa Fluor 633-conjugated transferrin was added to the cells. After fixation and permeabilization the cells were stained with primary antibody against ErbB2 (Sc08) and GM-488. Note that ErbB2 is internalized after geldanamycin (GA) treatment and colocalizes with transferrin (arrows). (B) After fixation and permeabilization the cells were stained with Sc08 and A561 against cathepsin D followed by GM-568 and GaR-633. Colocalization of internalized ErbB2 and cathepsin D is indicated with arrows. (C) Ultracryosections of SK-BR-3 cells treated with geldanamycin for 2 hours before fixation. Labeling with Sc08 followed by GM-10 shows ErbB2 in MVBs. (D)Ultracryosection of a geldanamycin-treated SK-BR-3 cell treated as in C. The section was first immunogold-labeled for cathepsin D (A561, PAG-5) and then for ErbB2 (Sc08, GM-10) as in C. Three MVBs are seen (M1-M3, M2 cut tangentially). In M1 and M3 double-labeling for cathepsin D and ErbB2 is seen. (E) Histogram of the r²_i values from SK-BR-3 cells either pre-treated with 200 nM bafilomycin or left untreated for 1 hour before both samples were incubated with 3 µM geldanamycin added for 4 hours. Cells were stained as described in Fig. 1A, and the statistical analysis done using a two-sided Mann-Whitney U-test. Bars, 20 µm (A,B); 200 nm (C,D).