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For the intracellular pMek staining, colonies were washed with PBS and dissociated to single cells with a 5 min trypsin (Life technologies) incubation. Trypsinization was stopped through addition of medium. Cells were disaggregated and pelleted, washed with PBS, and immediately fixed with 1.5% PFA (Roth). The cell mixture was incubated for 10 min at room temperature and subsequently centrifuged for 5 min at 500×g.

Cells were resuspended in ice-cold MeOH, incubated for 10 min on ice (0.5 ml/1 × 106 cells) and centrifuged for 5 min at 500×g. Cells were washed once with staining buffer (PBS + 1% BSA (Sigma), 2 ml/1 × 106 cells) and blocked for 10 min in staining buffer. Cells were incubated with the pMek-specific antibody (Cell Signaling, #2338, 1:100, antibodies are listed in Additional file 9: Table S8) for 30 min at room temperature (100 μl/1 × 106 cells), then washed twice with staining buffer. Cells were then incubated with an anti-rabbit-Alexa647 antibody (Thermo Fisher Scientific,1:400) for 15 min at room temperature (100 μl/1 × 106 cells), washed twice with staining buffer before FACS sorting using the BD FACSAria™ II.

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