Fig 4. The SPY992 biosensor closely tracks the expected kinetics of EGFR phosphorylation and displays increased specificity for EGFR pTyr⁹⁹² site of EGFR in live-cell imaging.

(A) Yeast cells displaying SPY992 on the cell surface were labeled with biotinylated synthetic peptides corresponding to the pTyr⁹⁹² or Tyr⁹⁹² sites in EGFR and the pTyr¹⁰²¹ site in PDGFR, followed by secondary labeling with SA-PE. A control sample of yeast cells labeled with only SA-PE is also shown. n = 3 independent experiments. (B) Normalized membrane recruitment of SPY992 in response to EGF stimulation of EGFR-expressing NR6 cells pretreated with gefitinib (n = 27 cells from 2 independent experiments) or not pretreated (n = 110 cells from 5 independent experiments), as measured by TIRF microscopy. p <0.0001 by two-way ANOVA. (C) Normalized membrane recruitment of SPY992 in response to EGF stimulation of NR6 cells expressing the c’1000 EGFR truncation mutant (n = 20 cells from 4 independent experiments) and NR6 cells expressing the c’1000F EGFR mutant with the Y992F mutation (n = 23 cells from 5 independent experiments). p <0.0001 by two-way ANOVA. (D) Normalized membrane recruitment of SPY992 in response to PDGF stimulation of NR6 parental cells (n = 21 cells from 4 independent experiments). (E) Normalized membrane recruitment of SPY992 in response to EGF stimulation of MDA-MB-231 cells pretreated with gefitinib for 5 min. (n = 97 cells from 4 independent experiments) or not pretreated (n = 52 cells from 6 independent experiments). p <0.0001 by two-way ANOVA. (F) Normalized membrane recruitment of SPY992 in EGFR-expressing NR6 cells in response to stimulation with a sub-threshold concentration of EGF (300 pM). Data from cells with an active response (n = 105 cells from 5 independent experiments) and no response (n = 95 cells from 5 independent experiments) are shown. p <0.0001 by two-way ANOVA. 95% confidence intervals are shaded (B–F).