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. 2022 Dec 15;122(2):301–309. doi: 10.1016/j.bpj.2022.12.014

Figure 4.

Figure 4

Evaluation of hetero-oligomer stoichiometry with progressive acceptor photobleaching. (A) YFP photobleaching abolishes FRET, dequenching mCer donor fluorescence. (B) Fluorescence microscopy of cells expressing Cer-ELN and YFP-SLN at 10-fold different concentrations. The dotted line delineates the margin of the low-expressing cell. (C) Progressive acceptor photobleaching revealed a linear donor/acceptor relationship for the low-expressing cell in (B) consistent with a hetero-dimer. (D) The high-expressing cell in (B) showed a curved donor/acceptor relationship consistent with a higher-order hetero-oligomer that contains more than one FRET acceptor. (E) As in (C) and (D). Data are mean ± SE for n = 10 cells. (F) The reciprocal experiment, with progressive acceptor photobleaching of cells expressing Cer-SLN and YFP-ELN, showed a linear donor/acceptor relationship consistent with a hetero-dimer. Data are mean ± SE for n = 10 cells.