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<p><span></span></p> <span></span><p><strong>Cell culture, reagents and plasmids</strong></p> <p>COS7 cells are grown in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C, 5% CO2. MG132 was purchased from Sigma Chemical (St. Louis, MO.). PcDNA4.1His/Max and PGEX6p-1 were purchased from Invitrogen and Pharmacia respectively. TBLR1 cDNA and a variety of truncated derivatives were amplified from PcDNA.TBLR1 plasmid using proof reading Taq polymerase. A truncated SMRT cDNA SMRT(1-300) containing the first 900 nucleotides of the message and encoding the amino terminal 300 a.a. of the 2473 a.a. protein was amplified from a human placenta cDNA library. This fragment contains the RD1 domain of SMRT but lacks RD2 and RD3. It also lacks the C-terminal ID domains as well as both SANT domains <a></a>[<a href="http://www.biomedcentral.com/1471-2121/7/31#B36">36</a>]. PCR products were cloned in frame into PcDNA4.1His/Max for mammalian expression or <em>in vitro </em>translation and cloned in frame into PGEX6p-1 plasmid for bacterial expression.</p> <p><strong>Northern analysis</strong></p> <p>Multiple Tissue Northern Blot™ membranes were purchased from Clontech. Hybridizations were performed with [<span>α</span><sup>32</sup>P]dATP labeled probes that also contained a modified dCTP to facilitate removal of the hybridized probe so that the blot can be hybridized repeatedly (Strip-ez™, Ambion Inc, Austin TX 78744).</p> <p><strong>Real Time Quantitative PCR (rtqPCR)</strong></p> <p>A panel of total RNA from human tissues (First Choice Human Total RNA) was purchased from Ambion, Inc. Real-time RT-PCR was performed using a SYBR Green PCR kit (SuperArray) and the iCycler Real-Time PCR Detection System (BioRad). PCR was conducted at 95°C for 5 min, followed by 40 cycles at 95°C for 60 seconds, 55°C for 30 seconds, and 72°C for 40 seconds. Data was collected at 72°C, and a melting curve analysis was done at the end of the cycling program to verify the quality of PCR products. In some experiments the PCR products were collected and electrophoresed through a 1.0% (W/V) agarose gel to confirm the product size. Except for those used to identify TBLR1<span>β</span>, the primers were purchased from Super Arrays Inc and had the characteristics listed in Table <a></a><a href="http://www.biomedcentral.com/1471-2121/7/31/table/T2">2</a>. The primer pair used to identify TBLR1<span>β</span> had the sequence: sense 3'-CTGCCTCACCATTTGGTTGT; 3'-ATCTGCAAAACCGTTGGAAA.</p> <p><strong>Preparation of cell lines ectopically expressing TBLR1</strong></p> <p>Jurkat cells were used to produce cells lines that stable expressed TBLR1 and its variants TBLR1, TBLR1 [<span>δ</span>N] and TBLR1 [<span>δ</span>C] constructs were made using proof reading PCR. Each sequence was amplified from templates with EcoR1 and Xho1 sites on the 5' and 3' ends. The amplified products were digested and the fragments were cloned into the MIG vector provided by Dr. David Baltimore. This vector contains an IRES site downstream of the cloning site and this is followed by a sequence encoding green fluorescent protein (GFP). Phoenix 293-Ampho cell line were used to produce the viruses. They were cultured in 10% FCS DMEM to 80% confluence and transfected with 3 <span>μ</span>g of each construct and 1 <span>μ</span>g of VSVg using Lipofectamine-2000 in the presence of 2 ug/ml of polybrene. The medium was changed at 24 hours. 72 hours after the transfection, the medium containing virus was collected and used to infect the Jurkat cells. Five days after infection, each Jurkat cell line was sorted for GFP positive cells. The sorted green cells were maintained in 10% FCS RPMI medium. The expression levels of the expressed proteins were confirmed by Western blotting.</p> <p><strong>Anti TBLR1 antibodies</strong></p> <p>To facilitate analyzing the expression of the TBLR1 proteins, the deduced peptide sequences were used to prepare anti-peptide antibodies directed against the non-identical portions of the two isoforms. Rabbits were immunized with the carboxyl terminal peptides of TBLR1<span>α</span> and TBLR1<span>β</span>. The deduced sequences of the C-terminal peptides are:</p> <p>TBLR1 <span>α</span> TQTGALVHSYRGTGGIFEV<strong>CWNA(C)AGDKVGASASDGSVCVLDLR</strong></p> <p>TBLR1 <span>β</span> TQV<strong>CLHYLNGQVLLNLGRSI</strong>CLYTLPHHLVVIPLVALIELLVLK</p> <p>The rabbits were immunized with the peptides shown in bold face above coupled to ovalbumin in Freund's Adjuvant (CFA) and boosted with the same antigens in Incomplete Adjuvant every 2 weeks for a total of 4 injections. Antibody titers were measured by ELISA using both the immunizing peptide and the recombinant TBLR1. Since the peptide used to produce the antibody for TBLR1<span>α</span> is identical to the homologous peptide found in TBL1, we also prepared an antibody directed against the region of TBLR1 (amino acids 117–125; <strong>AASQQGSAK</strong>) that differed most from TBL1. The resultant antibody was then absorbed with a peptide (CGVSHQNPSK-amide coupled to acrylamide) representing the equivalent sequence in TBL1, to further reduce cross-reactivity. The resultant antibody was then affinity purified using the immunizing peptide. The site recognized by this antibody is 30 amino acids downstream of the putative F-box in the amino terminal half of TBLR1.</p> <p><strong>Western blotting</strong></p> <p>1.0–2.0 mg of tissue or 10–20 million cells were lysed with SDS-lysis buffer and the lysates denatured at 100°C for 5 minutes. Samples were electrophoresed through either 10% SDS gels, transferred to nitrocellulose membranes, blocked with 5% non-fat milk and stained with the primary antibody at the dilution recommended by the distributor. The membrane was washed and stained with a secondary, horseradish peroxidase-labeled conjugate. After washing, the membranes were exposed to a detection cocktail containing hydrogen peroxide, phenol, and luminol and the light emitted by the HRP/hydrogen peroxide catalyzed oxidation of luminol, was detected by blue-light sensitive autoradiography film. In some experiments the TBLR1 proteins were isolated by immuno-affinity chromatography using the reagents provided by Pierce Chemical in their Seize Primary Immunoprecipitation kit. Rabbit anti-TBLR1(117-125) described above was covalently coupled to the gel. The bound material was eluted with a glycine-HCl buffer (pH 2.8) and then run on the gels.</p> <p><strong>Immunofluorescence</strong></p> <p>Mouse 3T3 cells were grown in DMEM with 10% fetal bovine serum. washed, fixed with 3.8% paraformaldehyde, permeabilized with 0.0.5% Triton-PBS and blocked with 10% human serum in PBS. They were then incubated with either anti-TBLR1 [117-125] or anti TBLR1<span>β</span> antibody (1:25) and stained with FITC-F(ab')2 anti-rabbit IgG antibody that had previously been absorbed to render them unreactive with human immunoglobulins (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA 19390). Normal rabbit serum was used as a control.</p> <p><strong>Cell cycle parameters</strong></p> <p>Cells in log phase growth were washed, resuspended in PBS and fixed in PBS paraformaldehyde (0.25%final). After 15 min at room temperature the cells were washed once with PBS and the pellet suspended in cold methano (-20°C)l. After 20 minuutes the cells were again washed aresuspended in PBS containing RNAse (100 ug/ml) After 20 minutes at 37°C the samples were resuspended in propidiumiodide (PI) at 10 ug/ml. The cells were analyzed after 90 minutes of staining.</p> <p><strong>Immunohistochemistry</strong></p> <p>Slide-mounted tissue sections are deparaffinized and hydrated through graded alcohols to running water. All slides are treated with 3°/v hydrogen peroxide in methanol for 30 min to remove endogenous peroxidase activity. All 3 antibodies were used at a final concentration of 1:25 and detected with HRP (horseradish peroxidase) coupled goat anti rabbit IgG that had been absorbed to reduce reactivity with human immunoglobulins (Jackson ImmunoResearch Laboratories). Normal rabbit serum was used as a control.</p> <p><strong>Co-immunoprecipitation</strong></p> <p>COS7 cells were transfected using lipofectamine (Gibco). After 48 hours, the cells were collected and proteins extracted with RIPA buffer. The protein extracts were incubated with the primary monoclonal antibody for 4 hours at 4°C and the resulting complexes bound to protein A/S agarose beads. After washing with RIPA buffer, the beads were resuspended in loading buffer and boiled for 5 minutes to elute the proteins. These proteins were used for Western blotting.</p> <p><strong>Growth arrest after transfection</strong></p> <p>HK293T cells were stained with 50 uM of the lipophilic carbocyanine, DiI (Molecular Probe cat# V-22885), in serum free DMEM for 8 minutes at 37°. After 3 washing with warm medium 200,000 cells were transferred to each well of a 6 well cluster plate. The cells were transfected with either an empty PCDNA4 control vector or PCDNA4 derived vectors containing TBLR1, TBLR1 [<span>δ</span>N] or TBLR1 [<span>δ</span>N] using Lipofectamine 2000 (Invitrogen). 4 <span>μ</span>g of plasmid was used for each transfection. After 48 and, 72 hours, the cells from each well were harvested and analyzed for the expression DiI. The data was analyzed using the "Proliferation Wizard" included in ModFit LT for Win32 (Verity Software House, Topsham, ME 04086).</p> <p><strong>Sequence analysis</strong></p> <p>Plasmid DNA was sequenced by the chain termination method in the Core Sequencing Facility of NYU Cancer Institute using an Applied Biosystems automated sequencer. The resulting sequences as well as the ESTs that were identified using the NCBI BLAST program were assembled using Sequencher software (Gene Codes Corp., Ann Arbor, MI) under a license to the Research Computing Resources of the NYU Medical Center.</p> <p><strong>GST pull-down experiment</strong></p> <p><sup>35</sup>S labeled proteins were synthesized <em>in vitro </em>using a T7 <em>in vitro </em>translation kit from Promega. GST and GST fusion proteins were induced with 0.1 mM IPTG and purified on glutathione-agarose column. To perform pull-down experiments, 10 ul of <sup>35</sup>S labeled proteins were mixed with 20 ul 50% agarose coupled with GST or GST fusion proteins in 150 ul PBS. The reaction was incubated with frequent rotation for 2 hrs at 4°C, and then washed extensively with PBS, containing 0.1% NP40. The beads were resuspended in SDS-PAGE loading buffer, boiled for 5 minutes to elute the bound proteins which were electrophoresed on 10% SDS-PAGE gel and visualized with a phosphorimager.</p> <p><br><strong>Competing interests</strong> <br><br>The author(s) declare that they have no competing interests. <br><br><br><strong>Authors' contributions</strong> <br><br>X-MZ isolated and sequenced TBLR1. He performed the pull-down experiments described here. LZ performed all of the real time PCR analyses. JG carried out the Western blots.SB did the sequence alignment and the homology analysis. CQ performed the cell cycle analysis and contributed to the design of some the experiments. RSB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. <br><br><br><strong>Acknowledgements</strong> <br><br>This work was supported by U.S. Public Health Service grants HL73713 and DK43376 from the National Institutes of Health and by the NYU Cancer Institute. Bioinformatics support was provided by the NYU School of Medicine Research Computing Resource, supported by National Science Foundation grant BIR-9318128. The Flow Cytometry Laboratory is supported by the NYU Cancer Center Core Grant (P30 CA 16097) The insightful comments of David Levy, Professor of Pathology, N.Y.U. School of Medicine are gratefully acknowledged. <br></p>
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