RNA concentrations were quantified by measurement of absorbance a

RNA concentrations were quantified by measurement of absorbance at 260 nm. The integrity of the cDNA was assessed with the Taqman gene expression assays, done on RPLPO housekeeping gene. Each Wortmannin ATM sample was normalized to the housekeeping gene levels. Mcl 1 primers were from Life Technologies. Cycle conditions for all PCRs were as follow an initial incubation of 2 min at 95 C followed by 35 cycles at 94 C 30 s, 55 C 30 s, 72 C 60 s. The amplification occurred for 2 min at 72 C. PCR products quantification was per formed as previously described in collaboration with Dr C. Asselin. Apoptosis assays Analysis of apoptosis was performed by quantification of the sub G1 peak by flow cytometry as previously described. Propidium iodide Inhibitors,Modulators,Libraries staining for DNA frag mentation was done by fixing cells and staining them with propidium iodide for DNA analysis content as pre viously described.

A total of 10,000 events were analyzed by flow cytometry and the percentage of hypo diploid cells was measured using a Inhibitors,Modulators,Libraries BD FACScalibur flow cytometer. Western blot analysis Cells were harvested and washed with ice cold PBS. Whole cell extracts Inhibitors,Modulators,Libraries were prepared in lysing buffer containing protease inhibitors and phosphatase inhibitors. Proteins were separated by 12% SDS PAGE gels. Proteins were trans ferred to PVDF membranes by electroblotting, and immunoblot analysis was performed as previously described. All primary antibodies were incubated overnight at 4 C in 5% fat free milk. Proteins were visualized by enhanced chemiluminescence. siRNA transfections The Fluorescein labeled Luciferase GL2 duplex or a non target siRNAs used as a control were from Dharmacon Research.

Cells were seeded in 6 well plates and allowed to adhere for 24 h. Cells were transfected with a mixture containing Lipofectamine Inhibitors,Modulators,Libraries 2000, opti MEM and siRNA. The siR NAs/Lipofectamine complex was then added to the media of 6 well plates containing cells. Cells were incubated for 4 6 h at 37 C in a CO2 incubator and medium containing FBS was then added. The Mcl 1 and FAK siRNAs were from Dharmacon Research, Akt siRNA from Cell Signal ing and Elk 1 siRNA from Santa Cruz. Immunohistochemistry staining TMAs were acquired from the Pan canadian platform for the development of biomarker driven subtype specific management of ovarian carcinoma. Sections were deparaffinized in citrate buffer containing 0.

05% Tween at 97 C for 20 min, washed with PBS and incubated with 3% peroxide. After treatment, slides were submerged in a citrate buffer for 15 min, and incubated with a protein blocking serum free reagent. The TMAs were stained by an immunoperoxidase Inhibitors,Modulators,Libraries method using an automated tissue immunostainer with Dorsomorphin ALK DABchromogen. The TMAs counter stained with hematoxilin and were visua lized by light microscopy at 20�� magnification and scored by two blinded independent observers using the H score method with an inter rating 90%. An intensity score of 0 3 was multiplied by the percentage of tumor cells stained to obtain the H score.

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