Fig. 2

RPPA analysis of T47D-RON cells shows robust phosphorylation of rpS6 in response to RON activation. a Clustered heatmap of expression of 305 proteins is shown for T47D-RON cells in MSP-dependent and -independent conditions and with or without the RON inhibitor ASLAN002. For MSP-dependent analysis, T47D-RON cells were treated with a low level of doxycycline (50 ng/ml) for 48 h, serum-starved for 24 h, and stimulated with MSP (100 ng/ml) for 30 min. For MSP-independent analysis, T47D-RON cells were treated with a high level of doxycycline (500 ng/ml) for 48 h ± RON inhibitor (ASLAN002) for 4 h. Lysates from three biological replicates for each group were sent for RPPA analysis. A cluster of proteins that are particularly RON sensitive are enlarged on the right. A second group of proteins differentially present in the “MSP-dependent” vs “MSP-independent” conditions was not affected by the presence of MSP or RON inhibitor, was enriched in proliferation and cell-cycle regulation genes, and was likely due to differences in serum levels between the conditions. These proteins are shown in Supplementary Figure 18. b Graph shows candidates whose phosphorylation or expression changed >1.5 fold in response to MSP. c–e Validation of RPPA results by Western blot analysis. RON activation, whether through MSP stimulation or RON overexpression, causes strong phosphorylation of rpS6 (both serines), p70S6K, and RSK, which can be reversed by RON inhibition using ASLAN002. GAPDH was used as loading control. Line indicates the separation between lanes on the same Western blots. f Schematic diagram showing differentially phosphorylated proteins upon RON activation in the context of the cellular signaling network^31,32,33,34