br DNA using the ReverTra Ace
DNA using the ReverTra Ace qPCR RT Kit (TOYOBO). Quantitative PCR was performed with SYBR Green Realtime PCR Master Mix (TOYOBO) and quantified by the Step One Real-Time PCR System (Applied Biosystems). Primers for real-time PCR are shown in Key Resources Table.
Immunoprecipitation (IP) and Western Blot
IP and Western blot were conducted as previously described (Liu et al., 2010a). Transfected HEK293T cells were lysed in CoIP lysis buffer (50 mM Tris-cl, pH 7.4, 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 10% glycerophosphate, and a cocktail of proteinase inhib-itors). After lysis for 30 mins, the soluble fraction of the cell lysates was isolated via centrifugation at 12,000 rpm in a microcentrifuge for 15 mins at 4 C. For IP, the cell lysates were centrifuged to remove the cell debris and were incubated in HA-conjugated beads (Abmart) or M2 beads (Sigma) for 2-3 hrs. Endogenous SPOP was immunoprecipitated using an anti-SPOP polyclonal antibody. The beads were boiled after extensive washing; The proteins were boiled, resolved via SDS-PAGE gel electrophoreses, and analyzed via immunoblotting. The proteins were detected using the Odyssey system (LI-COR Biosciences).
Cells were seeded on fibronectin-coated glass coverslips in 24-well tissue culture plates. After 24 hrs, the cells were rinsed once with PBS and fixed in 4% paraformaldehyde for 15 mins at room temperature. The fixed cells were permeabilized using 0.1% Triton X-100 and rinsed twice with PBS. The coverslips were blocked with blocking buffer for 1 hr (0.3% BSA in PBS) and incubated in a primary antibody in blocking buffer overnight at 4 C. Next, the coverslips were rinsed twice with blocking buffer and incubated in secondary 3-Methyladenine for 1 hr at room temperature in the dark, followed by tyramide signal amplification. The glass coverslips were mounted using Mowiol and were examined using a Zeiss LSM 510 Meta confocal system.
Biotinylated Peptides Pull-Down Assay
The biotinylated peptides were synthesized by GL Biochem (Shanghai) Ltd and dissolved in PBS solution. Briefly, 2 mg of peptides was preloaded onto 10 ml of Neutravidin agarose (Thermo). Then, 293T cells were transfected with HA-SPOP. 24 hrs post-transfection, cells were collected, lysed, and the lysates were incubated with the peptide-preloaded agarose for 3 hrs at 4 C in binding buffer (20 mM HEPES (pH7.9), 150 mM KCI, 1 mM DTT, 1 mM PMSF, 10% glycerol, 0.1% NP-40 and proteinase inhibitors). After five washes with washing buffer (20 mM HEPES (pH7.9), 150 mM KCI, 1 mM DTT, 1 mM PMSF, 0.1% NP-40, and pro-teinase inhibitors), the bound proteins were eluted by boiling in 2XLaemmli SDS loading buffer, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subjected to nitrocellulose membranes (Millipore), followed by western blotting analysis.
GST Pull-Down Assay
The His-NANOG protein was purified from E.coli and incubated in 10 mg of purified GST or GST-SPOP protein. The GST proteins were purified using glutathione sepharose 4B, and the bound His-NANOG was detected via Western blotting.
To test the specificity of pS68-NANOG antibody, the peptides (10 ng/ml) were coated to ELISA plate overnight at 4 C, Plates were washed followed by block at room temperature for 1 hr. After wash and incubated with the pS68-NANOG antibody and then Avidin-HRP*, the TMB solution was added to each well after wash, read plate at 450nm after Stop solution was added.
NANOG Peptide WT: DLLIQDSPDSSTSPKGKQP,
NANOG Peptide Ser68(P): DLLIQDSPD(p)SSTSPKGKQP,
NANOG Peptide S68A: DLLIQDSPDASTSPKGKQP.
Genome DNA Extraction and SPOP Sequencing
Genomic DNA from prostate tumor specimens was isolated using a QIAamp DNA Mini Kit (50) (Qiagen) and subjected to an initial pre-
PCR amplification step to enrich the SPOP exons 6, 7 and 8. Then SPOP mutation status was determined by Sanger sequencing.
SPOP primers (Exons 6, 7 and 8) were used for amplification and sequencing:
TCGA Data Analysis
Level 3 data for mRNA expression from TCGA were downloaded and processed using standard methods. Gene expression was analyzed using two-class unpaired significance analysis of microarrays (SAM) (http://statweb.stanford.edu/ tibs/SAM/)for the indi-cated tumors versus normal samples. Differences in expression were considered to be statistically significant when the fold change>2 and q<0.05.