Total RNA was isolated using the RNeasy mini kit according to the manufacturers instructions. selleck chemicals Axitinib Equal amounts of RNA were taken for cDNA synthesis using a High capacity cDNA Kit. Briefly, 2 �� reverse transcription master mix was prepared from 10 �� Inhibitors,Modulators,Libraries Reverse Transcription Buffer, 25 �� deoxyribonucleotide triphosphates, 10 �� random primers, and MultiScribe Reverse Transcriptase and mixed with equal parts of total RNA. The PCRs were per formed using TaqMan Gene Expression Assays using forward and reverse primers as well as internal probes Mm00839900 m1, Mm00489103 m1, Bcl w Mm00432054 m1 and BclxL Mm00437783 m1 for TWEAK, Fn14, Bcl w and Inhibitors,Modulators,Libraries Bcl xL, respectively. The PCRs were performed using 7500 Fast Real Time PCR System under the following condi tions, 50 C for 2 minutes, 95 C for Inhibitors,Modulators,Libraries 10 minutes, 40 cycles at 95 C for 15 seconds and 60 C for 1 minute.

Each experiment was Inhibitors,Modulators,Libraries repeated eight times. Determination of TWEAK and TNF a concentrations To determine the effect of hypoxia on the release of TWEAK from cerebral cortical neurons, we used an ELISA to quantify the concentration of TWEAK in the culture media of Wt neurons maintained under normoxic conditions or exposed to 0 to 360 minutes of OGD conditions. Each observation was repeated eight times. To measure the effect of TWEAK on the release of neuronal TNF a, Wt cerebral cortical neurons were incubated with TWEAK 100 ng mL or a comparable volume of vehicle, followed at 1, 5, 30 or 60 minutes by quantification of TNF a in the culture media with an ELISA kit following manufacturers instructions.

Each experiment was repeated with neurons cultured from three different animals, and each observation was repeated eight times. Western blot analysis Wt cerebral cortical neurons were incubated for 60 Inhibitors,Modulators,Libraries min utes with TWEAK 100 ng mL alone or in combination with the ERK 1 2 inhibitor 10 uM. After 0 to 180 min utes of incubation, cells were homogenized in radioim munoprecipitation assay lysis buffer, protein concentration was determined with the bicinchoninic acid protein assay and 16 ug of total protein were loaded for SDS PAGE elec trophoresis and immunoblotting with antibodies direc ted against pERK 1 2, total ERK 1 2, pBAD, total BAD and b actin. Each observation was repeated four to six times. Immunohistochemistry and determination of apoptotic cell death Wt mice received an intraperitoneal injection Nilotinib solubility of 0. 1 mL of TWEAK or a comparable volume of sal ine solution followed 24 hours later by tMCAO. Twenty four hours after tMCAO, brains were harvested and 10 um frozen sections were stained with the Apop Tag Plus Fluorecein In Situ Apoptosis Detection Kit fol lowing manufacturers instructions. Briefly, sections were fixed in 1% paraformaldehyde in PBS, pH 7.