Figure 7. Conformational equilibria and intrinsic affinity of intact α₅β₁ on cell surfaces.

Mean fluorescence intensity (MFI) after background subtraction is from quantitative fluorescence flow cytometry of K562 cells without washing.

1. Determination of EC₅₀ values for conformation‐selective Fabs from enhancement of 10 nM Alexa488‐Fn3_9–10 binding and fitting to Appendix Equation S2. Mean ± s.d. of least square fits to triplicates.
2. Affinity of α₅β₁ for Alexa488‐Fn3_9–10 in the presence of indicated Fabs.
3. Affinity of α₅β₁ for Fn3_9–10 by enhancement of 0.4 nM Alexa488‐12G10 Fab binding.
4. Affinities of α₅β₁ for Alexa488‐12G10 Fab in the presence of indicated Fabs.
5. Thermodynamics and intrinsic affinities of α₅β₁ conformational states on K562 cells and Jurkat cells (Appendix Fig S8). Affinity for Fn3_9–10 of the closed conformations of α₅β₁ on K562 cells was estimated from $K_{\mathrm{d}}^{\mathrm{EO}}$ using the same fold‐difference as found with Fn3_9–10 for the α₅β₁ ectodomain (Fig 4D). Since 12G10 stabilizes the open conformation only, thermodynamic calculations use K _a = 0 for the closed conformations.
6. Energy landscape plots, as described in the Fig 4E legend, comparing K562 α₅β₁ to the clasped and unclasped α₅β₁ ectodomain with complex glycosylation (Fig 5B).
7. Summary of the intrinsic affinities of α₅β₁ conformational states on K562 cells and comparison of the conformational equilibria for α₅β₁ on K562 cells and for the unclasped α₅β₁ ectodomain with complex glycosylation.

Data information: (B–F) Error definitions are as described in the legend to Fig 4.