The following antibodies were used: mouse monoclonal antibody (mAb) anti-TRα1/β1 (C-1), rabbit polyclonal antibodies anti GR (H-300), ERα(H-20), RXRα(D-20), RARα(C-20), PPARγ(H-100) (Santa Cruz Biotechnologies, Santa Cruz, CA); rabbit polyclonal antibody anti-TACC1 (Upstate cat # 07-229); mAb anti-Flag M2 and mAb anti-Flag M2 HRP (Sigma); anti rabbit and anti mouse IgG Horseradish peroxidase (HRP) conjugate (Promega); anti rabbit and anti mouse Cy3-conjugated IgG and anti rabbit fluorescein (FITC)-conjugated IgG (Jackson) and mouse and rabbit Trueblot (eBioscience).
Yeast two-hybrid screen
Experiments were performed according to the Clontech Laboratories protocols: manual PT3061-1, for the Matchmaker two-hybrid system 2 in yeast (catalog # K1604-1), and handbook PT3024-1 for the yeast protocols. The full length TRα2 to be used as bait was cloned from a cDNA library built from 17-day mouse embryos (Clontech ML4006AB) to be used as bait. TRα2 is a natural variant form of the thyroid hormone receptor encoded by the TR gene that is unable to bind the ligand T3, in contrast to the receptor TRα1. TRα2 was fused downstream to the GAL4-DBD in pAS2-1. Yeast cells, strains CG1945 and Y190, were transfected in parallel with the plasmid pAS2-1- TRα2 and were selected in the presence of 1 mM or 20 mM 3-AT (aminotriazole), respectively on an SD/-Trp medium. Yeast strains expressing the TRα2-GAL4 DBD were then transfected with a prey cDNA library constructed from 17-day mouse embryo cDNA fused to GAL4-AD in the pGAD10 vector. Transfected clones were selected on an SD/-Trp/-Leu/-His/+3-AT medium (1 mM or 20 mM depending on the strain, see above).
GST pulldown assay
The GST-TACC1-J fusion construct cloned into the pGEX-3X plasmid were produced in bacteria upon induction with 0.2 mM IPTG at 37°C. The bacteria were sonicated, the lysate was centrifuged and the supernatant incubated with glutathione-Sepharose 4B beads (Pharmacia) for 1 h at 4°C in PBS/0.1 mM PMSF/1% Triton buffer. Beads were washed and protein interactions were performed with [35S]-Met labelled TRα synthesised in vitro using TNT kits (Promega). The Sepharose beads were subsequently washed three times with the PBS/PMSF/Triton buffer. Bound proteins were eluted with loading buffer at 100°C and analysed by SDS-PAGE and fluorography (Amplify, Amersham).
Plasmids and constructs
Human TACC1 cDNAs were amplified from a cDNA library of activated RAJI cells by PCR with the following primers: forward 5'-AGATCTGAATTCCATGGCGTTCAGCCCGTGGC-3', reverse 5'-AGATCTGAATTCTCAGTCAGTCTTTCCCAGC-3'. The four PCR products were subcloned into the pGEM-T vector (Promega, Madison, Wis.) and sequenced. The human TACC1 cDNAs (TACC1-A,-K,-S, and -J) were then cloned into the BglII site of pSG5-Flag vector or pEGFP-C1.
Point mutations (TACC1 amino acids (AA) 609 (Leu) and 610 (Ile) (hTACC1 full-length protein numbering) were introduced using the commercial QuikChange Site-Directed Mutagenesis kit according to the manufacturer's protocol (Stratagene).
The pSG5 rat TRβ1 and pSG5 mouse TRα1 constructs were gifts from ML. Privalsky. The pSG5 rat TRα2 was provided by MA. Lazar. The pGL3 human ERα (hERα), and pSG5 mouse PPARγ constructs were gifts from V. Laudet. pSG5 GR was gift from JM. Vanacker. PSG5 mouse RXRα and RARα were gifts from H. Escriva. The luciferase reporters used in transfection experiments contain chicken E6 lysozyme Thyroid hormone Responsive Element (E6-TRE) from Lee and Privalsky and Retinoic Acid Response Element (RARE) from G. Benoit. Renilla Luciferase used for normalization was from Promega.
Truncated form of TRα1 (TRα1 Δ helix12) was amplified by PCR using High Fidelity polymerase (Roche) and oligonucleotides that introduce EcoRI sites on the 5' and 3' ends, followed by subcloning into the EcoRI site of the pSG5 vector.
All media were purchased from Invitrogen (Invitrogen/Gibco, Carlsbad, CA). HEK-293F, HeLa and COS-7 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 6% heat-inactivated fetal bovine serum, glutamine (2 mM), penicillin/streptomycin (1000 U/ml). F9 mouse embryonic carcinoma cells were cultured on 0.1% gelatin-coated plates in the medium described above but supplemented with MEM sodium pyruvate (1 mM). Cells were maintained at 37°C in the presence of 5% CO2. Whenever indicated, charcoal-stripped fetal bovine Serum (Biochrom, AG) was used and hormones were used at 10-6M or 10-7M.
Indirect immunofluorescence microscopy
Cells were seeded overnight on coverslips onto a 24 -well plate and transfected or not with 0.5-1 μg of DNA with Exgen500 (Fermentas protocol). 48 h after transfection, cells were fixed for 15 min in 0.5% formaldehyde/1.5% glutaraldehyde diluted in PBS. After extensive washing, cells were subsequently overlaid with the appropriate antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) diluted in 5% normal goat serum in the presence of 0.1% Triton X-100 for 1 h at room temperature. After washing, anti-mouse or rabbit Cy3 or FITC conjugated secondary antibody (Jackson ImmunoResearch Laboratories) was added and the cell nuclei were later counterstained with DAPI (4', 6-diamidino-2-phenylindole dihydrochloride hydrate) (Sigma), Propidium Iodide or DRAQ5 (Biostatus limited). The coverslips were mounted on microscopy slides with FluorSave embedding medium (Calbiochem) and imaged on the appropriate fluorescence microscope.
Cos-7 cells were transfected using lipofectamin according to the manufacturer's protocol (Invitrogen/Gibco, Carlsbad, CA). Cotransfected cell lysates were pre-cleared at 4°C for 2 h by adding 100 μl protein G-Sepharose bead slurry (50%) per ml (Amersham) or anti-Flag M2 affinity gel (Sigma), and 800 μg were incubated at 4 C for 2 h in the presence of the appropriate antibody (2 μg) or of a non specific control antibody (2 μg). The lysates were then incubated overnight at 4°C with 100 μl protein G-Sepharose (50% slurry) (Amersham) or anti-Flag M2 affinity gel (Sigma). The Sepharose was washed twice with RIPA buffer and three times with the washing buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton ×100) and immobilized proteins were eluted with 50 μl protein gel loading buffer. Immunoprecipitation was followed by Western blotting to detect bound proteins.
Western blot analysis
Proteins were separated by SDS-PAGE then transferred to nitrocellulose membranes, which were incubated for 2 hours at room temperature with blocking buffer [TBST (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) + 2% non-fat dry milk]. After extensive washing with TBST, the blots were hybridized for 1 hour with primary antibodies followed by 1 hour incubation at room temperature with HRP-conjugated secondary antibodies against mouse or rabbit IgG (Promega, Madison, Wis.) or true-blot detection system (eBioscience) accordingly. The proteins were detected using SuperSignal® West Pico Chemiluminescent, as recommended by the manufacturer (Pierce, Perbio) and autoradiographed.
Total proteins were extracted 48 h after transfection in RIPA buffer (50 mM Tris pH 8, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton ×100, 0.1% Na-deoxycholate) supplemented with Protease Inhibitor Cocktail (Complete, Roche). After homogenisation at 4°C for 20 min, samples were centrifuged at 10,000 × g for 15 min at 4°C and the supernatant containing proteins frozen and stored at -80°C. Nuclear and cytoplasmic proteins were isolated using Chemicon's Nuclear Extraction Kit according to the manufacturer's protocol.
For F9 subcellular fractionation, cells were collected in ice cold 1 × PBS and re-suspended at 4 × 107 cells/ml in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, and protease inhibitor cocktail [Roche]). Triton X-100 was added (0.1% final concentration), the cells were incubated on ice for 8 min, and the nuclei (fraction P1) were collected by centrifugation (5 min, 1,300 × g, 4°C). The supernatant (fraction S1) was clarified by high-speed centrifugation (5 min, 20,000 × g, 4°C), and the supernatant (fraction S2) collected. The P1 nuclei were washed once in buffer A and lysed for 30 min in buffer B [3 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol and protease inhibitor cocktail (Roche)]. Insoluble chromatin (fraction P3) and soluble (fraction S3) fractions were separated by centrifugation (5 min, 1,700 × g, 4°C). The P3 fraction was washed once with buffer B and re-suspended in SDS-Laemmli buffer and boiled for 10 min. Protein concentrations were determined by BCA protein assay (Interchim).
Synthetic double-strand siRNA specific for the human TACC1-J coding region (Custom Smart Pool siRNA) and the siRNA ON-TARGET plus siCONTROL non-targeting siRNA n°1 were purchased from Dharmacon (Perbio). The day before transfection, COS-7 and HEK-293F cells were seeded at 5 × 104 and 1.5 × 105 cells/ml, respectively. Cells were transfected with 50 nM of siRNA, using Dharmafect 1 (Dharmacon, Perbio) according to the manufacturer's protocol.
For transactivation-reporter assays, cells were seeded onto 24-well plates overnight and transfected with DNA and Exgen500 according to the manufacturer's protocol (Fermentas). Transfected cells in each well were lysed and processed for a dual luciferase assay according to the manufacturer's protocol (Dual-Luciferase Reporter Assay, Promega, Madison, Wis.). Luminescence was determined with a luminometer (Veritas, Microplate Luminometer, Turner Biosystems). Relative luciferase activity (RLU) was calculated as the Firefly luciferase activity normalized to Renilla luciferase activity from cotransfected pRL-CMV-luc.
For quantitative analysis real-time quantitative PCR was carried out using primers designed with the Primer 3 software and produced by MWG (see Additional file 5). The Mx3000p Detection system (Stratagene) was used with QuantiTect SYBR Green PCR Master Mix (Qiagen) for the detection of PCR products. The cycle threshold was set at a level where the exponential increase in PCR amplification was approximately equal between all samples.