Fig. 4. Geldanamycin treatment induces a redistribution of ErbB2 in the plasma membrane and increases receptor mobility. (A) Confocal images of live SK-BR-3 cells transfected with ErbB2-CFP before and after 1 hour of geldanamycin treatment. ErbB2 resides in protrusions of the plasma membrane before stimulation, but not after stimulation with geldanamycin. (B) Diagram showing the mobile fraction of ErbB2-CFP measured using FRAP before and after treatment with 3 µM geldanamycin for 15-60 minutes. The P-values are derived using a two-sided Student's t-test (n=42) and the error bars represent 95% confidence intervals. (C) Mobile fractions of ErbB2-CFP in SK-BR-3 cells treated with 3 µM geldanamycin for 1 hour. The cells were divided into two groups depending on their degree of internalized ErbB2 as described in the Materials and Methods. The P-value is derived using a two-sided Student's t-test (n=79) and the error bars represents 95% confidence intervals. Note that the cells with high internalization also exhibit a significantly higher mobile fraction. (D-G) Electron micrographs of SK-BR-3 cells immunogold labeled for ErbB2 on the cell surface (pre-embedding immunogold labeling). CP, clathrin-coated pits/profiles. (D) A control cell where ErbB2 is primarily associated with membrane protrusions. (E,F) The surface of two geldanamycin-treated cells where the ErbB2 distribution is more even on the surface of the cell. (G) The surface of a cell where ErbB2 has been crosslinked to antibody. ErbB2 is associated with the bulk membrane between protrusions including CPs. Bars, 20 µm (A); 200 nm (D-G).