Mouse or rabbit igg

Nuclear protein extracts from 15-day-old livers or cells were prepared as previously described^{31 (link)} using the high-salt extraction method (10 mM HEPES-KOH pH 7.9, 380 mM KCl, 3 mM MgCl₂, 0.2 mM EDTA, 20% glycerol, and protease inhibitors). For IP assays, nuclear lysates were diluted threefold by adding ice-cold HENG buffer (10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2, 0.25 mM EDTA, 20% glycerol) and precipitated with antibodies overnight at 4 ^oC followed by incubation for 2 h with protein G Sepharose beads (Millipore). Mouse or rabbit IgG (Santa Cruz) was used as a negative control. Immunoprecipitates were washed five times (10 mM HEPES-KOH pH7.9, 300 mM KCl, 0.3% NP40, 1.5 mM MgCl2, 0.25 mM EDTA, 20% glycerol and protease inhibitors), eluted and resolved on 10% SDS-PAGE. For bXAB2 and bXPF pulldowns, 0.6–0.7 mg of nuclear extracts were incubated with M-280 paramagnetic streptavidin beads (Invitrogen) as previously described^{31 (link)}.

In TRAMP PCa, immunostaining for apoptotic (cl-caspase-3, Cell Signaling Technology) and proliferating (Ki67, NCL-Ki67p, Leica Biosystems, Buffalo Grove, IL) cells was performed using rabbit polyclonal and biotinylated goat anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA) as previously described [50 (link)]. Blood vessel density was determined by immunostaining for CD31 using a goat polyclonal antibody (M20; Santa Cruz Biotechnology) and a biotinylated rabbit anti-goat secondary antibody. Immunostaining for Mcl-1 (S-19) was performed using a 1/50 dilution of rabbit polyclonal and biotinylated goat anti-rabbit IgG secondary antibody. Immunostaining for γH2AX (clone JBW301# 05-636; EMD Bioscience) was performed using a 1/200 dilution of mouse monoclonal and the Vector Mouse on Mouse Peroxidase Kit (Vector Laboratories) following the manufacturer's instructions. The number of cleaved caspase-3, Ki67, γH2AX positive cells and CD31 positive vessels were determined for 1198, BA, 1198 + BA, and vehicle controls (n=3-5 each group), as previously described [51 (link)]. For negative controls, we used the same concentration of mouse or rabbit IgG (Santa Cruz Biotechnology) instead of specific primary antibodies, resulting in lack of immunostaining.