However, we demonstrated that Roscov itine at doses preferentially inhibiting C

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 However, we demonstrated that Roscov itine at doses preferentially inhibiting C Empty However, we demonstrated that Roscov itine at doses preferentially inhibiting C

Post  jy9202 on Fri Apr 11, 2014 5:22 am

MUC1 cytoplasmic domain dimerization can be disrupted by an engineered Fv domain and a monomeric ligand To investigate the importance of MUC1 dimerization in Src association and ICAM 1 induced signalling, we manipulated dimerization using a chimeric construct of MUC1 and a C terminal Fv domain, which is FKBP with a F36V mutation, allowing for specific interaction between INNO-406 bcr-Abl 阻害剤 the engineered Fv domain and bivalent or monovalent ligands. Impor tantly, the Fv domain itself does not facilitate dimeriza tion of proteins, but following addition of Fv domain ligands, dimerization status can be manipulated.

Pre viously, this system has been used to successfully manip ulate dimerization of growth factor receptors and G protein coupled receptors, Mechanistically, the bivalent ligand, which contains two Fv binding domains, effectively brings two Fv domain containing proteins within close proximity dimerization, The monovalent ligand, Lapatinib EGFR 阻害剤 which contains one Fv domain binding domain, is designed to bind to Fv domain containing proteins and sterically inhibit their interaction with other pro teins disaggregation or monomerization, MUC1 CD dimers were stabilized after Fv ligand treatment by addition of the DSS cross linker prior to cell lysis.

We found that treatment of 293T MUC1 CFP Fv cells for one minute with increasing concentrations of AP20187D did not increase the quantity of MUC1 CD dimers above no treatment, while AP21998M treat ment resulted オーダー Lonafarnib in a dose dependant reduction in MUC1 CD dimerization, After treatment with 1 uM of monomerizing Fv ligand, AP21998M there was a 60% reduction in detectable MUC1 CD dimers, As a control, 293T MUC1 CFP cells, which lack the Fv domain, do not show a change in dimer quantity follow ing treatment with AP20187D or AP21998M, Densitometric analysis of the MUC1 CFP dimer band normalized to monomer band illustrates this observation further, MUC1 CD dimer disruption results in decreased recruitment of total and active Src kinase To determine the importance of MUC1 CD dimeriza tion in constitutive Src recruitment, 293T MUC1 CFP Fv, and, as a control, 293T MUC1 CFP cells were treated with increasing concen trations of AP20187D or AP21998M for one minute and immunoprecipitated with anti MUC1 CD.

Following separation on SDS PAGE, blots were probed with anti Src and anti SrcP416, In the MUC1 CFP Fv transfectants, the amount of total and active Src associated with MUC1 CD decreases in a dose depen dent manner with AP21998M treatment, Treatment with AP20187D did not result in a significant change, and 293T MUC1 CFP cells were unaffected by Fv ligand treatment. Densitometric analysis of Src and SrcP416 compared to MUC1 CD are given in Additional File 1. These data suggest that MUC1 CD dimers, but not monomers, con tain a recruitment, and potentially an activation, motif for Src kinase. MUC1 CD dimer disruption results in decreased ICAM 1 binding induced calcium oscillations and cell invasion To determine if MUC1 CD dimerization is important in previously observed ICAM 1 binding induced events, we assayed parental, 293T MUC1 CFP, and 293T MUC1 CFP Fv, and 293T MUC1 CFP Fv cells for ICAM 1 binding induced calcium oscillations, and invasion through an ICAM 1 positive monolayer after addition of the Fv ligands 1 uM AP20187D or 1 uM AP21998M and compared this to a no treatment control.

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