Equilibrium dissociation constant of EGFR pre-dimerization is determined using Co-II.

(A) Schematic representation of the KD measurement of EGFR homodimerization using Co-II. EGFR-mEos3.2 becomes co-immobilized only when interacting with the surface-immobilized SNAP-EGFR by an anti-SNAP antibody; otherwise, it remains in a mobile state. (B) Trajectory map of CF660R-labeled SNAP-EGFR and EGFR-mEos3.2 before and after anti-SNAP antibody treatment in the same single COS7 cell growing with 10% FBS. A total of 200 trajectories are shown in each trajectory map. Scale bar, 3 μm. (C) Diffusion-coefficient distribution of SNAP-EGFR and EGFR-mEos3.2 before (black line) and after anti-SNAP antibody treatment (red line). The immobilization criteria are presented as a blue dashed line. (D) The immobilized fractions of SNAP-EGFR and EGFR-mEos3.2 before and after anti-SNAP antibody treatment. (E) Fluorescence images of total expression and single-molecule–level expression of SNAP-EGFR. Scale bars, 5 μm and 2 μm, respectively. A fluorescence intensity profile of single SNAP-EGFR shows a single bleaching step. (F) KD analysis using a binding curve of prey EGFR to bait EGFR (y-axis) with respect to the density of the antibody-induced immobilized bait EGFR (x-axis). The bound/unbound ratio of the prey with respect to the density is shown (left inset) with a linear fit (red solid line) and a 95% confidence interval (red dashed lines). Scatchard plot for EGFR pre-homodimerization is shown (right inset). The KD was determined in DMEM supplemented with 10% FBS at 37 °C. Each dot indicates data obtained from individual cells. (G) KD of EGFR pre-homodimerization measured in various cell lines. The error bars represent the SEM at the single-cell level (n > 4). (H) A spatial KD map of EGFR pre-homodimerization and the log-normal distribution of the KD values obtained from different regions of plasma membrane in a single living cell. Scale bar, 5 μm. (I) The KD profiles obtained from the cross sections corresponding to the red dashed lines in panel H. (J) The box plots displaying the distributions of KD values obtained from periphery or center regions of each single cell. n = 10. *p t test). A.U., arbitrary unit; DMEM, Dulbecco's Modified Eagle Medium; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; mEos3.2, monomeric Eos fluorescent protein variant 3.2; SNAP, SNAP-tag.