Gelatin zymography Brain pericyte conditioned media were subjected to zymography in line with the manufacturers recommendations concentrated by Amicon Ultra centrifugal filter units, and then. Cells were fed every 2 3 days by changing medium. After 10-14 days in culture, floating cells and weakly attached cells of the mixed primary cultured cell layer were removed by vigorous shaking of the flask. Then, astrocytes at the bottom of the AT101 culture flask were trypsinized and seeded in to new culture flasks. The primary cultured astrocytes were preserved in one hundred thousand FBS/DMEM. They were grown in a humidified atmosphere of fifty CO2/95% air at 37 C. Cells in the second or third passage were used for experiments. Western blot analysis Brain pericytes, astrocytes and RBECs were incubated with or without different levels of TNF an at 37 C for the indicated time. When protein kinase inhibitors were applied, they were added 15 min ahead of the program of TNF a. To evaluate the expression of TNF a receptor 1 and TNF a receptor 2 among mind pericytes, astrocytes and RBECs, these cells were employed without TNF a treatment. The culture supernatants were collected and concentrated 60 collapse applying Amicon Ultra centrifugal filter devices. Cells were lysed and scraped in phosphoprotein lysis buffer Skin infection containing 1% phosphatase inhibitor cocktail 1, 1% phosphatase inhibitor cocktail 1% and 2 protease inhibitor cocktail. The total protein concentration in cell lysates was determined utilizing a BCA Protein assay kit. Equivalent amounts of protein from each sample were electrophoretically separated on 5 2006-2007 SDS polyacrylamide fits in, and then used in polyvinylidene difluoride membranes. Membranes were blocked with Blocking One or Blocking One P for phosphorylated proteins. Phosphorylation of p42/p44 mitogen-activated protein kinase, p38 MAPK, d Jun N final kinase and Akt were detected with major antibodies against phospho p38 MAPK, phospho p42/p44 MAPK, phospho JNK and phospho Akt. MMP 9 and MMP 2 in tradition supernatant were detected using antibodies Anacetrapib against MMP 9 and MMP 2. TNFR2 and tnfr1 in cell lysates were detected with an anti MMP 9 antibody and anti MMP 2 antibody. After cleanup, membranes were incubated with an ideal horseradish peroxidase conjugated secondary antibody. Membranes were incubated in stripping buffer for 15 min twice, to reprobe Akt, p38 MAPK, JNK and whole p42/p44 MAPK. Full p42/p44 MAPK, p38 MAPK, JNK and Akt were found using main antibodies against p42/p44 MAPK, p38 MAPK, JNK and Akt. The immunoreactive bands were visualized using an ECL Advance Western Blotting Detection Kit. The band images were digitally taken with a FluorChem SP imaging process and band intensities were quantified using AlphaEaseFC pc software. The relative intensity of phosphorylation of individual proteins was expressed as the ratio of the corresponding total protein and phosphorylated protein.