Figure 1.

Decrease of FRET efficiency and acceptor fluorescence during FSAB. (A) Determination of the fraction of homoclustered ErbB1 and ErbB2 using FSAB. A431 cells were labeled with Cy5-Mab528 (●) or with a mixture of Cy5-Mab528 and AlexaFluor546-Mab528 (○). SKBR-3 cells were labeled with Cy5-trastuzumab (▾) or with a mixture of Cy5-trastuzumab and AlexaFluor546-trastuzumab (▵), and the samples were photobleached in the presence of CBr₄ at 543 nm. The directly excited acceptor fluorescence intensities are shown in the graph. Error bars indicating the mean ± standard error (SE) are shown for every third data point for clarity. (B) Determination of the fraction of heteroclustered ErbB1 in the absence and presence of EGF stimulation. Quiescent and EGF-stimulated SKBR-3 cells were labeled with a mixture of AlexaFluor546-trastuzumab and Cy5-Mab528 against ErbB2 and ErbB1, respectively. Nonsensitized, directly excited acceptor fluorescence and the FRET efficiency were calculated by recording fluorescence images in the donor, FRET, and acceptor channels when the bleaching illumination was interrupted at the excitation wavelength of the donor (□, directly excited acceptor intensity of nonstimulated cells; ○, FRET efficiency of nonstimulated cells; ▪, directly excited acceptor intensity of EGF-stimulated cells; ●, FRET efficiency of EGF-stimulated cells). Photobleaching was carried out in the presence of CBr₄. Error bars indicate the mean ± SE of ∼100 cells. (C) Determination of the fraction of heteroclustered ErbB2 in the absence and presence of EGF stimulation. SKBR-3 cells were labeled with a mixture of AlexaFluor546-Mab528 and Cy5-trastuzumab. Otherwise, the experimental conditions and symbol assignments are the same as in B.