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Our results reinforce the idea that it is necessary to develop new compounds th

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Our results reinforce the idea that it is necessary to develop new compounds th

Сообщение  jy9202 в Чт Авг 14, 2014 4:00 pm

In con trast, Rictor deficient cells showed a similar chemotactic response as control cells towards PDGF BB, indicating ARQ 197 c-Met 阻害剤 that mTORC2 is not involved in PDGF BB dependent cell migration, this is surprising since mTORC2 has been shown to regulate cell polarity and the dynamics of the actin cytoskeleton, although no alterations in the actin cytoskeleton were observed in Rictor null MEFs. Similarly, inhibition of mTORC1 and 2 in NIH3T3 cells did not influence the chemotactic properties of these cells. mTORC2 may affect cell migration by pro moting PKC dependent phosphorylation of the focal adhesion component paxillin. However, it has previ ously been found that PDGF BB can promote paxillin phosphorylation through the JNK MAP kinase pathway, and this may relieve the absolute requirement of mTORC2 in PDGF BB mediated fibroblast migration.<br><br> Conclusions The pathway from AZD0530 Sr 阻害剤 PDGFR leading to phosphorylation of Akt involves both the mTORC2 and PLCγ PKC path ways. In contrast, phosphorylation of S6 downstream of mTORC1 depends on PLD activation, but is independ ent of mTORC2 and Akt signaling. During conditions where Erk1 2 signaling is inhibited, the initial S6 phosphorylation is delayed. Interfering with mTOR signaling did not affect PDGF BB induced Erk1 2 phos phorylation. Functionally, inhibition of mTORC1 and 2 by rapamycin effectively blocked PDGF BB mediated cell proliferation. Figure 6 depicts a schematic figure of key roles of mTOR in PDGF BB induced cell signaling. Materials and methods Reagents Recombinant human PDGF BB was generously provided by Amgen.<br><br> The inhibitors CI 1040, triciribine and NVP BKM120 were from Calbiochem, Cayman Chemical Company and Selleckchem, respectively. Antibodies against phosphorylated Akt, phosphorylated mTOR, phosphory lated S6, cleaved caspase 3, phosphory lated Erk1 2 and phospho MARCKS were purchased purchase Alvocidib from Cell Signaling Technology. A B actin antibody was purchased from Sigma. A rabbit antiserum recognizing Erk was raised against a peptide corresponding to the carboxyl terminal sequence EETARFQPGYRS conjugated to KLH. The wild type control and Rictor knockout mouse em bryonic fibroblasts have been described previously and were kindly provided by Dr Mark Magnuson. PLCγ1 null MEFs have been described previously and were kindly provided by Dr Matilda Katan.<br><br> Cell culture The murine embryonic fibroblast cell line NIH3T3, and MEFs were cultured in Dulbeccos modified Eagles medium with 10% bovine serum, 100 U ml penicillin and 100 ug ml streptomycin. For serum star vation, cells were washed once and incubated in medium containing 0. 1% FBS. Lipase inactive PLC 1 H335F H380F, porcine aortic endothelial cells were cultured in Hams F 12 containing 10% bovine serum albumin, in the presence or absence of 20 ng ml doxycycline to induce protein expression. Immunoblotting Subconfluent cells were starved and incubated with ve hicle or inhibitors at the indicated concentrations and thereafter stimulated with PDGF BB for the indicated periods of time. Cells were washed two times in ice cold phosphate buffered saline and lysed in 20 mM Tris pH 7. 4, 150 mM NaCl, 5 mM EDTA, 1% Triton X 100, 0.


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