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Relative gene copy number analysis by quantitative real-time PCR. Quantitative real-time PCR for gene copy number measurement was performed on an ABI PRISM 7300 sequence detection system (Applied Biosystems) by using the Power SYBR Green PCR Master Mix (Applied Biosystems). For relative quantification of copy number of Akap12 and Cited2 genes, we used beta-2-microglobulin (B2m) as an internal reference gene. For the determination of a reference gene, we confirmed that B2m had a correlation coefficient of 0.80 with beta-glucuronidase (Gusb) in tumor DNA samples. The oligonucleotide sequence and product size used are shown in Supplementary Table 1.
Immunoblotting. Cell lysates from OB and frozen SB-207499 of OS were prepared by using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, 1 mM sodium orthovanadate, and 1 mM NaF) supplemented with 0.2 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Complete EDTA-free, Roche Diagnostics). Equal amounts of protein were fractionated by 7.5–10% SDS–PAGE, transferred to nitrocellulose membranes, and reacted with antibodies against SSeCKS (S1562; BD Sigma–Aldrich), Cited2 (sc-25375; Santa Cruz Biotechnology) and β-actin (AC-74; Sigma–Aldrich). The protein bands were visualized by enhanced chemiluminescence using ECL Plus Western blotting detection system (GE Healthcare).