Combination therapy with sorafenib and AZD6244 for 3 h resulted in inhibition of Erk and Ret activites at low concentations that was preserved for both the cell lines, consistent with the complete in the MTT assay. the level of phospho Erk was paid off start at levels of 0. 1 uM in both the cell lines since 1 h after treating the cells, but phosphorylated Erk was detectable after 3 h of treatment and levels returned to pre ubiquitin conjugating exposure levels after 6 h despite continuous exposure to the compound. Erk activation was totally inhibited at 0. 5 uM dosing in both the cell lines. The total Erk appearance remained the same during all the treatments. As predicted, western blots after everolimus treatment show merely a significant decrease in phospho p70S6K, a direct downstream goal of mTOR, and AZD6244 induced a significant decrease in phospho Erk beginning at levels of 1 uM without inhibiting other pathways. While both the substances elicited a rise in quantities of serine 473 phosphorylated Akt, Ret phosphorylation was also induced by everolimus. Taken together, the data suggest that at doses below the cell viability IC50, sorafenib only transiently inhibited Erk phosphorylation, suggesting Posttranslational modification that preservation of this inhibition may be beneficial in increasing the natural effects of this compound. In addition they claim that the relative resistance to everolimus and AZD6244 as solitary agents may include activation of Ret or Akt. Sorafenib is synergistic with AZD6244 in both the cell lines, other combinations were nonsynergistic To determine, whether the western blot analysis of sorafenib therapy predicted synergy, mix studies were performed using concentrations of sorafenib below and at the cell viability IC50 for both the cell lines. In these studies, combination of low dose sorafenib along with amounts of AZD6244 below its individual IC50 caused somewhat greater inhibition of chk2 inhibitor TT and MZ CRC 1 cell expansion compared with either agent alone that has been synergistic on statistical analysis. The synergistic effect was less pronounced within the MZ CRC 1 cell line and only became cytotoxic at higher levels. By comparison, the mixture of everolimus and sorafenib didn’t generate considerably greater inhibition of TT and MZ CRC 1 cell development compared with either agent alone. Also, everolimus and AZD6244 combination treatment wasn’t synergistic. These data suggest that loss of Erk inhibition may be responsible simply for the loss of sorafenib effect at low doses and that this is often exploited with therapeutic intent for combination therapies. Combination therapy signaling Next, we wanted to make sure the combination therapies were inhibiting the targets by western blot.