Fig. 6. CTX-induced effects in the association properties and biological functions of ErbB2. (A) The homoassociation of ErbB2, and its heteroassociation with ErbB1 and ErbB3 was measured with flow cytometry on control cells (black bars) and cells pretreated with 8 µg/ml CTX-B at 37°C for 30 minutes (white bars). Error bars indicate the standard error of the mean calculated from three independent experiments (^*P<0.01). (B) SKBR-3 cells were seeded in 6-well plates and starved for 48 hours. CTX pretreatment was carried out in the presence of 8 µg/ml CTX-B at 37°C for 30 minutes, and then cells were stimulated with EGF or heregulin for 10 minutes. The tyrosine phosphorylation of ErbB2 (left graph) and Shc (right graph) was measured in control (black bars), EGF-stimulated (white bars) and heregulin-stimulated (right hatched bars) samples. Membranes labeled with anti-phosphotyrosine antibody were stripped and reprobed with anti-ErbB2 or anti-Shc antibody. The phosphotyrosine content of the bands was normalized to the amount of protein (ErbB2 or Shc) immunoprecipitated and to the control samples. Asterisks indicate statistical significance of Student's t-test performed on non-stimulated and growth factor-stimulated samples (^*P<0.1, ^**P<0.05). Error bars indicate the standard error of the mean calculated from three independent measurements. (C) Control () and CTX-pretreated () cells were incubated with unlabeled 4D5 antibody for 40 minutes. Samples were taken every 10 minutes, and cell surface ErbB2 was labeled with Alexa488-7C2. The fluorescence intensity of Alexa488-7C2-labeled cells was measured with flow cytometry. Error bars indicate the standard error of the mean (n=3). (D) Control and CTX-pretreated SKBR-3 cells were incubated with Alexa488-4D5 antibody at 37°C for 30 minutes. Cells were imaged using confocal microscopy, and vertical slices of the cells are shown. The approximate position of the cell membrane is marked with a black dashed line (bar, 1 µm). (E) 50,000 cells were seeded in culture dishes. Control SKBR-3 cells and cells treated with 4D5, CTX-B and both with 4D5 and CTX-B were cultured for 5 days. Treatment with the antibody and CTX-B was carried out as described in Materials and Methods. At day 5 the cell surface expression of ErbB2 was measured with flow cytometry by labeling cells with Alexa488-7C2. The black bars show the mean fluorescence intensity after subtraction of autofluorescence. The number of cells is shown by the white bars. Error bars indicate the standard error of the mean (^*P<0.05 compared with the corresponding control, n=3).

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