Iap family antibody sampler kit

Cells were seeded in 6 well plates at a density of 0.4x10 6 /well, treated with poly(I:C) and cisplatin for 45 minutes and irradiated. Proteins were isolated after 24 hours by resuspension of the cells in RIPA buffer containing protease inhibitor cocktail (Complete Mini EDTA-free, Roche.) and the cells were sonicated. 4× Laemmly buffer (0.25 M Tris-HCl pH 6.8, 20% DTT, 8% Na dodecil sulfate, 40% glicerol, 0.008% bromphenol blue) was added to obtain 1× mixture and the samples were heated to 95˚C. For Western blot analysis equal amounts of proteins (40 µg) were separated on 10% or 12% SDS-PAGE and transferred onto 0.2 µm nitrocellulose membrane (BioRad). The membranes were blocked with 5% nonfat dry milk and were stained with primary antibodies: IAP Family Antibody Sampler Kit (#9770, Cell Signaling Technology)), PARP (Calbiochem) and actin (Sigma-Aldrich). Afterwards, the membranes were stained with peroxidase conjugated secondary antibody (NA934V, Amersham, in a concentration 1:3000) and visualized with the chemiluminescent system (Perkin Elmer).
Densitometric calculations of the band intensities corresponding to specific protein were performed using Uvidoc Cambridge Chemiluminescence Imaging system (Uvitec Cambridge) and UVIband software (Uvitec Cambridge).