Analysis of Downstream Signaling of TLRs after U0126 Inhibition

Francisella tularensis (F. tularensis) is a pathogenic bacterium which causes the disease tularemia. F. tularensis is thought to be able to enter and multiply inside of liver cells because it interacts with toll-like receptors (TLRs). Examining the signaling pathway downstream of the TLRs will allow for better understanding of how this disease works and possible treatment strategies. In this study I propose to examine how molecular inhibitors affect signaling downstream of TLRs in HepG2 cells treated with lipopolysaccharides (LPS) from E. coli. Mitogen-activated protein kinase is a cellular molecule in signaling cascades that directs cellular response mechanisms. MAPK can be activated by LPS. The signaling pathway will be examined for the ability of UO126 to block the activation of MAPK. The ability of UO126 to block MAPK will be examined with SDS-polyacrylamide gel electrophoresis and a western blot. Phospho-MEK antibodies will be utilized during the western blot. It is predicted that kinase inhibitors, such as UO126, will cause a decrease in downstream signaling after mock-infection. A decrease in downstream signaling will potentially cause a decrease in cellular response to the infection