Reference 46th Indicated causes synergistic combination judged by the analysis of apoptotic cells in the G1 population of cells by PI flow cytometry Fnd Rbt .. 3605 of the cell cycle, or 5% BSA. Antique Rperbindung Gamma Secretase cancer was visualized by verst Markets chemiluminescence using the SuperSignal West Dura or Pico reagents from Pierce. For the treatment FastapTM alkaline phosphatase, tumor were crushed or lysed in a buffer with phosphatase inhibitors or in lysis buffer without inhibitors. They were then treated with either mock or AP, respectively for 1 h at 37th The reaction was stopped by heat inactivation at 75 and by the addition of 10 mM sodium orthovanadate in the lysis buffer. The samples were then separated on SDS-PAGE and transferred to nitrocellulose membranes.
Immunofluorescence. Briefly, cells in MeOH at 20 after 1 h and then fixed in saline Solution blocked with phosphatase 10% FCS and 0.1% saponin. The samples were then incubated for 16 h at 4 with tubulin antibody Body. 488 anti-mouse secondary Ren DyLight LY2940680 F Staining was for 1 h at the 37th The cells were treated with PI-cons found Rabbit and mounted for microscopic analysis using a standard protocol cytospin. RNA-Pr Para tion and quantitative analysis by reverse transcription-PCR. RNA from cultured cells was performed using RNA II NucleoSpin. cDNA synthesis was 1 g of RNA using a kit iScript first strand synthesis performed. qRT PCR was addressed using KAPA SYBR FAST qPCR kit, cDNA and primers against ODC, performed CHEK2, Myc and ubiquitin on a machine IQ real-time PCR.
The relative mRNA levels were measured using the methods DDCT. Mouse experiments. All animal experiments were performed in accordance with the approval of the regional animal ethics committee conducted A6 # 08 or # 08 A18. The p53 knockout M And APCmin mice, both C57BL / 6 background were obtained from Jackson Laboratory. λ Myc Mice were a kind gift from Dr. Georg Bornkamm. All transgenic Mice were t Possible for signs of disease. All moribund Mice were immediately get Tet. In tumor-bearing Mice get Follow-up, tumors and lymphoid organs were Collected for the analyzes or tissue banks. The tumors were either frozen low as parts and / or dispersed into single cell suspensions of scalpels and wires snapping cells.
For determining the Transplantationskompabilit t lymphoma, were receiver nozzles singer of C57BL / 6 M Intravenously through Se injection of 500,000 cells that are either injected against a shRNA CHEK2 or a non-targeting vector and then monitored for tumor progression. If the lymphoma was observed palpable, were Mice get tet, And tumor material was frozen snap-blot analysis of protein gel. A bone marrow Myc p53-deficient entered to develop Born in vivo model, the magnetically sorted from B cells followed by labeling with anti-B220 Antique Body and anti-R PE PE magnetic microbeads, by loading on a MACS-S Column from. Purified B cells were added 4 overnight in RPMI1640 medium containing 10% FCS, 2 mM L-glutamine 50 M mercaptoethanol, 0.1875% sodium bicarbonate and antibiotics in the presence of a retrovirus Myc MSCV IRES GFP, prepared as above, and g / ml polybrene described.
Infected cells were injected into C57BL / 6, and tumor development was monitored and in a medium containing 10% DMSO frozen banking.62 MEDCHEM the axon. FastapTM alkaline phosphatase was purchased from Fermentas. Cell culture. 293T human kidney cells and NIH 3T3 fibroblasts were f from ATCC and cultured in Dulbecco, modified Eagle’s medium containing 10% Fetal K Calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotics purchased. Mouse lymphoma cell lines that were of tumors originating in transgenic M Nozzles λ Myc in a density of 105 cells / ml in RPMI 1640 medium containing 5% FCS, 2 mM L-glutamine, 50 M mercaptoethanol, bicarbonate bred 0.1875% of sodium and antibiotics. Mouse embryo fibroblasts from E13.5 embryos were temporally E15 pairing between M Nnchen and female heterozygous p53 generated by previous methods