Protein extraction For making whole cell lysates, the cells were lysed in radioimmune precipitation assay buffer supple mented with protease inhibitor cocktail. Nuclear and cytoplasmic fractionations were performed with Proteo JET Cytoplasmic and Nuclear Protein Extrac tion Kit according to manufac turers protocol. Western blot merely analysis Equal amounts of cytoplasmic, nuclear, or whole cell extracts were electrophoresed on sodium dodecyl sul fate polyacrylamide gels, and then transferred onto a polyvinylidene difluoride membrane. The transformed membrane was blocked for 1 h and incu bated with indicated primary antibodies at 4 C overnight. The primary antibodies usedwere as follows, rabbit anti iNOS, b actin, p65, Lamin B, I B a, ERK1 2, p38, JNK and mouse anti phosphorylated ERK1 2, p38, JNK antibody.
The membrane was washed three times with Tris bufffered saline containing 0. 05% Tween 20 for 10 min and incubated with anti rabbit or anti mouse IgG horseradish peroxidase at room temperature for 1 h. The Supersignal West Pico chemi luminescent substrate system was used to detect immunoreactive bands. Inhibitors,Modulators,Libraries The intensity of protein bands after western blotting were quantitated by using Quan tity One Version 4. 6. 3 Image software and normalized against proper loading controls. Electrophoretic mobility shift assay Nuclear extracts were prepared as described above. Oli gonucleotides corresponding Inhibitors,Modulators,Libraries to the binding site con sensus sequences were synthesized and end labeled with biotin by Invitrogen. EMSAs were performed using the LightShift Inhibitors,Modulators,Libraries chemiluminescent EMSA kit.
Briefly, 20 fmol of biotin labeled, double strand probe was incu bated for 20 min at room temperature in 20 ul of EMSA binding buffer Inhibitors,Modulators,Libraries containing 2. 5% glycerol, 5 mM MgCl2, 50 ng ul poly, 0. 05% Nonidet P 40, and 6 ug of nuclear proteins. For competition EMSA, 200 fold excess unlabeled, double strand probe was added to the binding reaction. The DNA nuclear pro tein complexes were resolved by electrophoresis in 6% nondenaturing polyacrylamide gel in 0. 5 �� Tris borate EDTA buffer at 100 V. Gels were then electro blotted onto Hybond nylon membranes at 380 mA for 50 min. The membranes were then cross linked for 15 min with the membrane face down on a transilluminator at 312 nm, and the biotinylated protein DNA bands were detected with HRP conjugated streptavidin using the chemiluminescent nucleic acid detection system.
Statistical analysis Data are expressed as means SEM of the indicated number of independent experiments. Changes in I B protein Inhibitors,Modulators,Libraries levels were analyzed by two our site way ANOVA. All other data were analyzed by one way ANOVA. Least significant difference post hoc test was used for multiple comparisons. Statistical analysis was performed using the SPSS software version 17. 0. P 0. 05 was consid ered statistically significant.