For Cer–Ven dimers, we obtained FRET efficiencies of roughly 0.55,
0.38, and 0.05 for linker lengths 5, 32, and 228 respectively, matching
previous work^30. The relative proportion of Cer and Ven fluorophores
in each cell was determined as NCer = Cerdirect/(1 − ED) and NVe n = Vendirect/
(gVe n/gCer × fVe n/fCer). To construct FRET two-hybrid-binding curves, we
imposed a 1:1 binding isotherm as in previous studies^30. For each FRET
pair, we obtained effective dissociation constant (Kd,EFF), ED,max and 95%
confidence intervals by constrained least-squares fit.
For Fig. 4b, c and Extended Data Fig. 9a, b, we show all individual
cells from two different transfections. We fit these data with the equa-
tion ED = Emax × [Ven − Rad]free/([Ven − Rad]free + Kd,EFF) using the least-
squares algorithm (https://www.mathworks.com/help/stats/nlinfit.
html). The Kd,EFF fit value based on all of the data points is shown as
the bar in Extended Data Fig. 9c. The error is calculated by the fitting
algorithm as a 95% confidence interval on the fit parameter (https://
http://www.mathworks.com/help/stats/nlparci.html)..)
Statistical analysis
Experiments were not randomized. Results are presented as
mean ± s.e.m. For comparisons between two groups, we used Student’s
t-test. Statistical analyses were performed using Prism 8 (Graphpad
Software). For multiple group comparisons, we performed a one-way
ANOVA followed by either Dunnett’s or Tukey’s post-hoc test using
Prism 8. Differences were considered statistically significant at values
of P < 0.05.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data and material availability
All transgenic mice are available from S.O.M. under a material agree-
ment with Columbia University. All data are available in the main text,
the Extended Data or the Supplementary Information. Proteomics
raw data and search results were deposited in the PRIDE archive and
can be accessed via the ProteomeXchange under accession numbers
PXD014499, PXD014500 and PXD014501. The FRET software is accessible
on github at https://github.com/manubenjohny/FACS_FRET. Source data
for Figs. 1 – 4 and Extended Data Figs. 1, 2, 6, 8 are provided with the paper.
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Acknowledgements We thank A. Karlin for helpful discussions and editing the manuscript;
B. Soda for creating the cell-model illustration; and G. A. Bradshaw for technical assistance.
This publication was supported by the National Institutes of Health (NIH; grants R01 HL113136,
R01 HL121253 and R01 HL146149) and by the National Center for Advancing Translational
Sciences (grant UL1TR001873). These studies used the resources of the Herbert Irving
Comprehensive Cancer Center Flow Cytometry Shared Resources, funded in part through
Center Grant P30CA013696. Images were collected (and analysed) in the Confocal and
Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center
at Columbia University, supported by NIH grant P30 CA013696 (National Cancer Institute).
A.P. was supported by NIH grant T32 HL120826 and National Science Foundation (NSF)
Division of Graduate Education (DGE) grant 1644869. D.R. was supported by grants T32
HL120826 and F31 HL142178. J.K. was supported by grant T32 HL007343 and the Glorney–
Raisbeck Fellowship from the New York Academy of Medicine, and J.A.H. was supported by
grant T32 HL007854. The content is solely the responsibility of the authors and does not
necessarily represent the official views of the NIH.Author contributions The following authors designed research and analysed data: G.L., A.P.,
A.N.K., S.I.Z., D.R., J.A.H., J.K., L.Y., B.-X.C., A.K, S.P.G., G.S.P., H.M.C., M.B.-J., M.K. and S.O.M. The
following authors performed research and analysed data: G.L., A.P., A.N.K., S.I.Z., D.R., J.A.H.,
J.K., L.Y., B.-X.C., A.K, K.D., G.S.P., H.M.C., M.B.J., M.K. and S.O.M. The following authors wrote
the paper with input from all authors: S.O.M., M.K., H.M.C., G.S.P. and M.B.J. All authors
provided feedback and agreed on the final manuscript.Competing interests The authors declare no competing interests.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
1947-z.
Correspondence and requests for materials should be addressed to M.K. or S.O.M.
Peer review information Nature thanks Donald Bers, Alice Ting and the other, anonymous,
reviewer(s) for their contribution to the peer review of this work.
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