Cell lines and treatment We employed H4 human neuroglioma cells in the experiments. The cells were cultured in DMEM containing inhibitor Tipifarnib 9% heat inactivated fetal calf serum, 100 unitsml penicillin, 100 ugml streptomycin, and 2 mM L glutamine. The cells were treated with 21% O2, 5% CO2 and 4% sevoflurane for two or six hours, as described by Dong et al. 21% O2, 5% CO2 and 4% sevoflurane were delivered from an anesthesia machine to a sealed plastic box in a 37 C incubator containing six well plates seeded with one million cells in 1. 5 ml cell culture media. A Datex infrared gas analyzer was used to continuously monitor the con centrations of delivered carbon dioxide, oxygen, and sevoflurane as performed in our previous studies. Harvest of brain tissues and cells, and protein level quantification Following the anesthesia, the mice were killed by decapi tation at P8.

The brain tissues were harvested and sub jected to Western blot analysis. The H4 cells were harvested in the end of the sevoflurane treatment or control condition. The harvested brain tissues and H4 cells were Inhibitors,Modulators,Libraries homogenized on ice using immunoprecipita tion buffer plus protease inhibi tors. The lysates were collected, centrifuged at 12,000 rpm for 10 minutes, and quantified for total Inhibitors,Modulators,Libraries pro teins with bicinchoninic acid protein assay kit. Signaling Technology, 9271. Finally, the antibody to detect non targeted protein B Actin was used to control for loading differences in total protein amounts. Western blot quan tification was performed as described by Zhang et al. Briefly, signal intensity was analyzed using image analysis program Quantity One.

We quantified the Western blots in two steps, Inhibitors,Modulators,Libraries first using B Actin levels to normalize protein levels and con trol for loading differences in the total protein amount. Second, we presented protein level changes in mice undergoing sevoflurane anesthesia as a percentage of those in the control condition. 100% of protein level changes refer to control levels for the purpose of com parison to experimental conditions. Statistics Data were expressed as mean standard deviation. Each group had 6 mice or wells of cells. We performed a power analysis based on our previous studies, and found that a sample size of 6 per arm would lead to a 90% power to detect a difference in the behavioral Inhibitors,Modulators,Libraries changes using a two sided t test with 5% type I error.

Given the presence of background AKTGSK3B activa tion in cells and brain tissues of mice, we did not use ab solute values to describe these changes. Instead, these changes were presented as percentages Inhibitors,Modulators,Libraries of those from the control group. For example, one hundred percent of AKT refers to the control level for the purpose of com parison to experimental conditions. Students t test was used to determine the difference between the sevoflur ane anesthesia selleck compound and control condition in the levels of P GSK3B and P AKT. P values less than 0. 05 were consid ered statistically significant.