Figure 2.

Validation of the IMPs using Single-Cell FRET Imaging

(A) Serum-starved Cos7 cells expressing Akt-PH-YFP (pseudocolored green) were stimulated with EGF, fixed, and stained for tyrosine-phosphorylated GIV (anti-pY1764GIV; red) and analyzed by confocal microscopy.

(B) Serum-starved Cos7 cells expressing IMP-Y1764, Y1798, or their corresponding YF mutants were stimulated with EGF and analyzed by confocal live-cell FRET imaging. Representative freeze-frame images from Videos S1, S2, S3, and S4 are shown.

(C) Time traces display the dynamic changes in FRET efficiency in (B); 12–15 cells were analyzed in 3 independent assays.

(D) Serum-starved Cos7 cells expressing IMP-Y1764 or the YF mutant were stimulated with LPA and analyzed by confocal live-cell FRET imaging. Representative freeze-frame images from Videos S5 and S6 are shown.

(E) Time traces display the dynamic changes in FRET efficiency in 12–15 cells that were analyzed in 3 independent assays.

(F) Immunoblots showing the contribution of serum (10%) on the phosphorylation status of wild-type (WT)-IMP-1764 and WT-IMP-1798 probes expressed in Cos7 cells at steady state. Phosphorylation of the IMP peptides and Akt was exclusively observed in fed state (10% FBS), but not in starved (0% FBS) conditions.

(G) Representative steady-state FRET images of Cos7 cells expressing IMP-1764 probe and its phosphorylation triggered by 10% serum. Higher FRET efficiency signals were detected in cells with 10% serum, but not in cells without serum.

(H) Scatterplots comparing the FRET efficiency at the PM in (G). Results are expressed as mean ± SD.

(I) Lysates of control and GIV-depleted MDA-MB-231 cells were analyzed for GIV depletion by immunoblotting (IB). The efficiency of depletion was estimated as ∼85% by band densitometry.

(J) Representative steady-state FRET images of MDA-MB-231 cells in (I) expressing the IMP-1764 probe.

(K) Scatterplots comparing the FRET efficiency at the PM in (J). Results are expressed as mean ± SD.