Western blot analysis Co cultured U87 cells, primary astrocytes or EAE brain tissues were homogenized in lysis buffer, and allowed to swell on ice for 30 min. Cell lysates were subjected to 8 10% sodium dodecyl sulfate kinase inhibitor Bosutinib polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were washed with phosphate buffered saline containing 0. 1% Tween 20, and then blocked for 1 h in PBST containing 5% skim milk. After washing the mem branes with PBST, the membranes were treated with antibodies against actin, CD40, CD40L, PKC isoforms, ERK, JNK, p38, Jak1 2, STAT1, CBP and TNFR1, and then mem branes were treated with p PKC isoforms, p ERK, p JNK, p p38, p JAK1 2, p ser727 STAT1, p Tyr diluted in PBST, and incubated for 60 min at room temperature.
Membranes were washed with PBST, and treated with HRP conjugated goat anti Inhibitors,Modulators,Libraries mouse or HRP conjugated rabbit anti goat IgG in PBST for 60 min. After washing, the protein bands were visualized using electrogenerated chemiluminescent solution. Electrophoretic mobility shift assay EMSA was performed with 32P labed probes and 2 ug of nuclear extract in 40 uL of EMSA reaction buffer. To perform the competition assay, a 100 fold excess of unlabeled competitor primer was added to the EMSA reaction mixture. Nuclear extracts were prepared from co cultured cells. Cells were washed twice with ice cold PBS, and resuspended in 1 ml ice cold buffer A. After incuba tion on ice for 15 min, the cells were lysed by adding Nonidet P40 and immediately vortexed for 10 sec. Nuclei were harvested by centrifugation at 20,000 �� g for 1 min and resuspended in 40 ul ice cold buffer C.
After incubation at 4 C for 20 min on a shaking platform, the nuclei were clarified Inhibitors,Modulators,Libraries by centrifuga tion at 15,000 �� g for 10 min. The supernatant was then Inhibitors,Modulators,Libraries transferred to a new tube, and quanti fied using Bradfords method. The 10 ul of a mixture of NF B oligonucleotide, T4 polynucleotide kinase 10 �� buffer, ATP, nuclear free water, and T4 polynucleo tide kinase were incubated for 30 min at 37 C. The reaction was stopped by adding 1 ul EDTA. After adding 89 ul Tris EDTA buffer, unincorporated nucleotides were removed from the DNA probe by chromatography through a G 25 spin column. The nuclear Inhibitors,Modulators,Libraries extract and gel shift binding 5�� buffer were incubated at room tempera ture for 10 min. Next, 20 30 fmol of 32P labeled NF B oligonucleotide was added, and the solution was incu bated at room temperature for 20 min. After incubation, Inhibitors,Modulators,Libraries 1 ul of 10 �� gel loading buffer was added to each reac tion. Reaction mixtures were electrophoresed on 6% polyacrylamide gels, and gels were analyzed Idelalisib clinical trial using FLA 2000.