The antibody against p ERK1 2 was utilized in Western blotting to detect the dual phosphorylation of ERK. The results showed that inside five minutes soon after incorporating EGF to cell culture medium, phosphorylation of ERK1 2 in AGS cells greater radically plus the phosphorylation was inhibited by pre infecting the cells with Ad PKG II and activating the enzyme with 8 pCPT cGMP. Activation of ras. Small G protein Ras is one more important element in MAPK ERK mediated signal pathway. It has two types in cells GTP bound energetic kind and GDP bound inactive kind. As soon as Ras is in GTP bound type, it might bind and activate Raf 1 and begin the consequent activations of serine threonine kinases within the signal pathway. We utilized pull down technique to detect the activated Ras. The outcome showed that immediately after incorporating EGF on the culture medium, active Ras in AGS cells increased clearly within five minutes.
Infecting the cells with Ad PKG II and stimulating them with eight pCPT cGMP ahead of incorporating EGF substantially prevented the EGF induced Ras activation. PKG II Inhibits EGF induced Activation of RAC1 Small G protein RAC1 is definitely the main member of Rho family which play important part in regulating migration of cancer cells. Both PLCc1 and MAPK ERK mediated signal transduction can activate RAC1 and thereafter stimulate PCI-34051 clinical trial cell migration. To even more confirm the inhibitory effect of PKG II on EGF EGFR induced signaling which is connected to migration, Pull down technique was applied to detect the inhibition of PKG II on activation of RAC1. The outcome showed that EGF remedy brought on an obvious grow of lively RAC1 and high action of PKG II effectively inhibited the activation of RAC1. This provided even further evidence within the inhibition of PKG II on EGF induced migration of gastric cancer cells.
get more information PKGII Interacts with EGFR and Brings about Thr phoshporylation on the Receptor The mechanism via which PKGII blocks the EGF induced activation of EGFR was preliminarily investigated in this experiment. Co Immunoprecipitation was applied to detect the interaction among PKGII and EGFR. Western blotting with pan anti phosphorylation of threonine antibody was employed to detect the Threonine phosphorylation of EGFR induced by PKGII. The results of Co Immunoprecipitation showed that in AGS cells contaminated with Ad PKG II and stimulated with eight CPT cGMP, direct binding involving PKG II and EGFR was detected. Success of Western blotting showed that activation of PKG II caused threonine phosphorylation of EGFR. This indicated that PKG II blocked the activation EGFR via binding with all the receptor and triggering phosphorylation of it. Discussion Now, two cGMP dependent protein kinases, PKG I and PKG II, have been identified in mammalian cells.