FIG 5.

Determination of the dissociation coefficients of Fos-Jun heterodimers and Fos-Fos homodimers from flow cytometric FRET titrations. (A, C) FRET efficiency measured in cells cotransfected with Fos²¹⁵-EGFP–Jun-mRFP1 and Jun-EGFP–Fos²¹⁵-mRFP1 and plotted as a function of the donor-tagged Fos²¹⁵ or Jun concentration. Data were grouped according to acceptor-to-donor molecular ratios (N_A/N_D) and fitted as described elsewhere (see equation S12 in the supplemental material) (solid lines), yielding the K_d value of the heterodimers and the FRET efficiency (E₀) of individual donor-acceptor pairs. Endogenous Fos and Jun were also taken into account. (B, D) The solid lines represent the maximal theoretically attainable E values at different N_A/N_D ratios (assuming E₀ values of 15 and 14.1% based on the fits) when all available Jun-mRFP1 molecules are engaged in heterodimers with Fos; the marked points correspond to the experimental N_A/N_D ratios (see equation S13 in the supplemental material). (E) Dependence of the K_d values from the fits on the Fos/Jun ratio. (F) FRET efficiency of Fos²¹⁵-EGFP–Fos²¹⁵-mRFP1 homodimers as a function of the donor-tagged Fos²¹⁵ concentration with K_d and E₀ yielded from a linked fit (see equation S14 in the supplemental material). (G) Maximal attainable FRET efficiencies at different N_A/N_D ratios (assuming an E₀ value of 9.47% based on the fit) when all Fos molecules form homodimers (see equation S15 in the supplemental material).