Ro can distinguish ERK1/2 phosphorylation in human xenograft tumors from surrounding mouse stromal cells in response to erlotinib treatment HCC827 xenograft mice have been treated with one particular dose of either water or erlotinib, and xenograft samples have been analyzed. Western blot evaluation indicated that EGFR Y1068 was completely dephosphorylated upon erlotinib therapy, whereas ERK1/2 was only partly dephosphorylated (Figure 2A). On assaying ERK phosphorylation with NanoPro, we observed two extra signals, pI values of five.24 and five.60, in HCC827 xenograft samples that were not present in HCC827 in vitro cultured cells (Figure 2B). These two peaks have decrease pI than the ppERK1 and pERK1 peaks observed in HCC827 cells. Because the theoretical pI worth of mouse ERK1 is decrease than that of human ERK1 (6.15 and 6.28, respectively, for non-phospho ERK1), we predicted that these two peaks are mouse ERK1 isoforms. Further analysis of mouse lung and skin samples confirmed the identity on the pI five.24 and pI five.60 peaks to become mouse ppERK1 and pERK1, respectively (Figure 2B). We also observed that, in erlotinib treated mouse xenografts, the human phospho-ERK1 signals decreased substantially, whereas the mouse phospho-ERK1 signals decreased only modestly (Figure 2C and 2D).LCS-1 Further evaluation with the lung and skin tissue samples from mice treated with erlotinib showed no important decrease in mouse lung or only modest decrease of ERK phosphorylation in mouse skin, when when compared with tissue samples from mice treated with water only (Figure S1A and S1B). NanoPro evaluation data indicate that the residual phospho-ERK activities observed in western blot have been derived from mouse stromal cells inNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMol Cancer Ther.ML115 Author manuscript; readily available in PMC 2014 November 01.PMID:26895888 Chen et al.Pagethe xenograft as an alternative to from human cancer cells. These information demonstrate that NanoPro technologies is able to distinguish human cancer cell-specific signals and their response to drug treatment from interfering mouse stromal cells in xenografts, and clearly revealed that erlotinib effectively inhibited down-stream Erk phosphorylation in targeted tumor cells but not surrounding stromal cells. Particular target response pattern detected by NanoPro in response to MEK inhibitor therapy Drug remedy of NSCLC cells with PD325901, an allosteric MEK1/2 inhibitor, resulted in dephosphorylation of ERK1/2, up-regulation of MEK1/2 pS218/S222 in HCC827 cells, and slight down-regulation of MEK2 pT394 in H2122 cells as observed in western evaluation (Figure S2A). Using NanoPro, we confirmed the drug inhibition on the phosphorylation of ERK isoforms (Figure S2B). Though HCC827 and H2122 cells exhibited diverse MEK1/2 peak profiles in un-treated baseline samples, a equivalent drug response signature was shared by both cell lines when treated with PD325901. By way of example, in comparison with H2122 cells, untreated HCC827 cells presented comparatively larger MEK1 pS218 signal at pI six.09 (examine Figure 3A 3E with 3B 3F), and MEK2 pS222 signal at pI five.98 (compare Figure 3C 3E with 3D 3F) plus a relatively decrease MEK2 pT394 signal at pI five.92 (examine Figure 3C 3G with 3D 3H). However with PD325901 treatment, as highlighted by arrows in Figure 3, distinct MEK response patterns appeared in each cell lines. The pattern was composed of a rise of MEK1 pS218/pS222 (pI five.60, five.91, and five.98 peaks) and MEK2 (pT394 pI 5.58) peaks; in addition to a lower of MEK.