Fig 2: Crosstalk between EGFR and RON is EGF-driven.(A and B) HEKRON/EGFR or A431RON cells were treated with ± 5 nM MSP or 50 nM EGF for 5 min at 37 °C. Representative immunoblots showing PY1068 and EGFR on cell lysates (A) or pan-phosphotyrosine (PY) and RON on samples immunoprecipitated (IP) with anti-HA antibody (B). (C and D) A431RON cells were stimulated with ± 20 nM EGF, 20 nM MSP or both for 5 min at 37 °C and immunoblotted as in (A and B). Triplicate biological experiments are quantified in the bar graphs to the right, shown as mean ± SD. (E) Representative immunoblots of a phosphorylation time course for A431RON cells treated with 5 nM EGF or 5 nM MSP and immunoblotted as in (A and B). Graphed values (right) are from triplicate biological experiments, normalized to maximal activation, and presented as mean ± SD. * p < 0.05; ** p < 0.01. Figure 1—source data 1.Full raw western blots and blots with relevant bands labeled, corresponding to Figure 1A, B, C, D and E. Figure 1—source data 2.Source data for quantification of blots in Figure 1C, D, and E.

Fig 3: Detection of RON and EGFR in human cancer cell lines with low, endogenous expression of both receptors.HCT116 colorectal carcinoma cells, SKBR3 breast cancer cells and A549 lung carcinoma cells were cultured overnight and then stimulated with 50 nM EGF or 5 nM MSP for 5 min. (A) RON receptor was IP’d with an anti-RON antibody and phosphorylated proteins in the IP were detected using a pY99 antibody. Here, detection was with HRP-antibodies. (B) Total cell lysates were run on a western blot and phosphoEGFR (pY1068) and total EGFR were detected. (C) Two-color detection using fluorescently-labeled antibodies for western using HCT116 cells. The phosphorylated protein does not overlap with full-length RON in the IP samples. The phosphorylated protein could be a truncated RON isoform or a different protein that co-Ips with RON in these cells. (D) The phospho-EGFR band does overlap with the full-length EGFR band.