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. 2020 Apr 15;11:1848. doi: 10.1038/s41467-020-15687-x

Fig. 4. Multiplexing the kinase activities of Akt, Src, and ERK in single live cells utilizing three biosensors containing stagRFP.

Fig. 4

a The red-far-red biosensor fRR-EKARev could reliably monitor ERK activity in response to 100 ng/mL huEGF stimulation in the red-far-red region, shown as changes in normalized emission ratio (far-red/red) (WT, n = 12 biologically independent cells; and pretreatment with 20 µM U0126, n = 5). b The yellow-red Akt activity reporter incorporating stagRFP reliably detects Akt activity in NIH 3T3 cells in response to 50 ng/mL PDGF stimulation, shown as changes in normalized emission ratio (red/yellow) (WT, n = 6; and pretreatment with 3 µM MK2206, n = 8). c The green-red Src activity reporter incorporating stagRFP reliably detects Src activity in Cos7 cells in response to 100 ng/mL huEGF stimulation, shown as changes in normalized ratio (red/green) (green for T-sapphire emission) (WT, n = 6; and pretreatment with 10 µM PP2, n = 12). The mean ratio ± SEM is displayed for a–c. d Multiplexed imaging revealed a potential kinetic relationship between Src, Akt, and ERK upon 50 ng/mL PDGF stimulation. Representative curves showing PDGF-induced responses of green-red based Src activity biosensor (blue), yellow-red based Akt activity biosensor (red), and ERK activity biosensor (green) in control cells without pretreatment (left), cells pretreated with 10 µM PP2 (middle), or 10 µM Src-I1 (right).