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Fig. 5. Cleavage of the ErbB2 kinase domain is not needed for internalization. (A-D). Confocal images of SK-BR-3 cells stained for ErbB2 as described in Fig. 1A. (A) ErbB2Cterminal staining dislocates from ErbB2total and is diffusely localized in the cytoplasm after 3 µM geldanamycin treatment, suggesting cytoplasmic cleavage of the receptor. (B) In addition, detection of the C-terminus of ErbB2 using another antibody (Ab-1) also showed similar dislocation from ErbB2total after 3 µM geldanamycin treatment for 4 hours, supporting cleavage of ErbB2. (C) In SK-BR-3 cells transfected with ErbB2-CFP and exposed to 3 µM geldanamycin for 4 hours, the C-terminal CFP-tag (detected using an antibody against the fluorophore) is also dislocated from ErbB2total after geldanamycin treatment. (D) ErbB2 is both internalized and cleaved (seen as diffuse blue staining, indicated with *) after 3 µM geldanamycin treatment. The enlarged region and purple structures show that both the C- and N-termini of ErbB2 are internalized. (E) A biotinylation assay detecting internalized ErbB2. Cells were treated with 30 µM geldanamycin for 3 hours at 37°C, Sc08 1:100 for 1 hour followed by G
M 1:100 for 1 hour at 37°C, or left untreated for 3 hours at 4 or 37°C. Both geldanamycin and crosslinking cause increased internalization of ErbB2 compared to the control situation but had no effect on the transferrin receptor (TfR). Note that in contrast to the other experiments we have used a tenfold increased concentration of geldanamycin. (F,G) Ultracryosections of SK-BR-3 cells treated with 3 µM geldanamycin for 2 hours before fixation. C-terminal ErbB2 was labeled with Ab-1 followed by PAG-5 (arrows), and then N-terminal ErbB2 with Sc08 followed by GM-10 (arrowheads) on the cell surface (F) as well as in a MVB (G). Bars, 20 µm (A-D); 200 nm (F,G).
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