The normal DNA. The values obtained were as homozygous loss, loss of heterozygosity, amplification Rkung and amplifier Rkung classified. Materials The following GDC-0879 antique body were used: anti-pERK1 / 2, anti-ERK, and the fight against vinculin from Sigma, anti-AKT Becton Dickinson, anti-PACT, PSRC anti, anti-PMET, anti-phosphorylated signal transducer and activator of transcription 3, anti- , pPaxillin pp130CAS and anti-technology cell signaling, Src anti-anti-p70 S6 kinase, S6 kinase, anti-PP70, and the fight against a Src homology 2-Dom ne-containing proteins from the transformation of Upstate Biotechnology, anti-CCND1 Dako, anti-Met, anti-STAT3, anti-CRAF, anti-phosphorylated focal adhesion kinase, the fight against FAK, anti PSHC and antiactin from Santa Cruz Biotechnology, anti-paxillin from Transduction Laboratories, anti-p130CAS from Abcam, anti-breast cancer resistance protein and fight against multidrug resistance protein 4 from Monosan, the anti-Kit of MBL, and peroxidase-conjugated secondary Ren anti-mouse immunoglobulin and anti-rabbit immunoglobulin G were used.
Immunpr Zipitation with anti-phosphorylated tyrosine and MALDI-TOF mass Dacinostat NVP-LAQ824 spectrometry analysis, the samples were processed as described above. Only proteins Were identified in at least three separate experiments tested. PLX4032 was in agreement with Plexxikon SU11274/Sugen, Inc., UO126, PHA 665752, BMS 354 825 / dasatinib, JNJ 38,877,605, SGX 523, and bought E804/Indirubin were obtained. After dose-response studies, the drugs were used at the indicated concentrations.
Routes the data analysis can be set using linear regression with four parameters dose-response sigma Concerning von and IC 50 values for inhibition of cell growth to 72 hours of treatment Gt PLX4032 were calculated using Prism software v. 5.0. Student were followed using St-test and ANOVA by Bonferroni correction to assess statistical significance. Interaction was assessed as elsewhere, with interaction index values gr He shows a synergy as described. The reported data are repr Sentative for three independent Independent experiments. Results effects of growth inhibitors in PLX4032 BRAFV600E mutated melanoma cells are not compatible with other common genetic defects melanoma including normal loss of PTEN connected the growth inhibitory effect of PLX4032 has been on a panel characterized of 27 genetically tested melanoma cell lines were 20 lines heterozygous for The V600E BRAF mutation and 7 lines of wild-type gene showed.
The effect of other genetic changes Confinement Lich mutations in CDKN2A, PTEN and p53 and amplification of tumor BRAF and MITF, pulled the sensitivity of melanoma cells with PLX4032 considered. It was established that PLX4032 strictly dependent inhibition of cell growth Ngig by the presence of BRAFV600E and regardless of other cotes Changes is. In fact, were 18 of 20 BRAFV600E mutated melanoma cell lines sensitive to this compound with IC50 values in the range between 0.01 and 1 M, w Displayed during two cell lines low sensitivity, and showed IC 50 values, which were about 10 M . The different IC 50 values were not mutational patterns of cell lines Lich Including the BRAF gene amplification or MITF, or associates of the expression of KIT protein. Melanoma cell line, LM20 and LM38 was prime Re resistance to PLX4032