For statistical analy sis, the number of cells with morphological features of apop totic cell death was determined by scoring IAP signaling pathway 100 cells from 20 different microscopic fields. The cell pellet was http://www.selleckchem.com/products/carfilzomib-pr-171.html resuspended in 400 l hypotonic lysis buffer containing pro tease inhibitors and was incubated on ice for 15 minutes. Then 12. 5 l of 10% NP 40 were added and the cell suspension was vigorously mixed for 15 seconds. The extracts selleck chemicals were cen trifuged for 1. 5 minutes. The supernatants were frozen at 70 C. Then 25 l ice cold nuclear extraction buffer were added to the pellets and incubated for 30 minutes with intermittent mixing. Extracts were centrifuged and the supernatant transferred to pre chilled tubes for storage at 70 C. Western blot analysis To determine the effect of resveratrolcurcumin on IL 1 dependent IBphosphorylation, IBdegradation and p65 translocation, whole cell lysates, cytoplasmic and nuclear extracts of chondrocyte monolayers were prepared and frac tioned by SDS PAGE. The total protein concentra tion of whole cell, nuclear and cytoplasmic extracts was determined using the bicinchoninic acid assay system using BSA as a stand ard. Equal quantities of total proteins were separated by SDS PAGE under reducing conditions. The separated proteins were transferred onto nitrocellulose membranes. Membranes were pre incubated in blocking buffer skimmed milk powder in PBS0. 1% Tween 20 for 1 hour, and were incubated with primary antibodies against p65, IB, p IB, VEGF, Cox 2, MMP 3, MMP 9, active caspase 3, PARP, Bcl 2, Bcl xL, TRAF1, collagen type II, Sox 9 and Actin. Membranes were washed three times with blocking buffer, and were incubated with alkaline phosphatase conjugated secondary antibodies for 30 minutes. They were finally washed three times in 0. 1 M Tris, pH 9. 5, containing 0. 05 M MgCl2 and 0. 1 M NaCl. Nitrob lue tetrazolium and 5 bromo 4 chloro 3 indoylphosphate were used as sub strates to reveal alkaline phosphatase conjugated specific antigen antibody complexes. The IKK complex was immunoprecipitated from whole cell lysates with antibodies against IKK and IKK and sub sequently incubated with protein AG agarose beads. After 2 hours of incubation, the beads were washed with lysis buffer and resuspended in a kinase assay solution containing 50 mM HEPES, 20 mM MgCl2, 2 mM dithiothreitol, 10 M unlabelled ATP and 2 mg substrate GST IB, and were incubated at 30 C for 30 minutes.
This was followed by boiling in SDS PAGE sample buffer for 5 minutes. The proteins were transferred to a nitrocellulose membrane after SDS PAGE under reducing conditions as described above. Phosphorylation of GST IBwas assessed using a specific antibody against phospho specific IB. To dem onstrate the total amounts of IKK and IKK in each sample, whole cell lysates were transferred to a nitrocellulose mem brane after SDS PAGE under reducing conditions as described above. Detection of IKK and IKK was performed by immunoblotting with either anti IKK or anti IKK antibod ies. Statistical analysis The results are expressed as the means standard deviation of a representative experiment performed in triplicate. The means were compared using Students t test assuming equal variances.