3112 Nucleic Acids Research, 2009, Vol. 37, No. 9 and MCF7 were performed 24 h after seeding cells using FuGENE 6 according to the manufacturer’s instructions (Roche, Sussex, UK). Cells were maintained in phenol red-free media with 5% charcoal-stripped serum 48 h before experimentation. The CK1d isoform was silenced using two independent siRNAs, targeting the following sequences: 50-ccggtct aggatcgaaatgtt-30 and 50-ctccctgacga ttccactgta-30. At 48 h (or 72 h) post-transfection, cells were treated with either vehicle (ethanol) or E2, and harvested later (as indicated) for RNA or protein analyses. RNA isolation and quantitative RT-PCR Isolation of total RNA was performed using the RNeasy kit (Qiagen). RNA (1 mg) was reverse transcribed using oligo (deoxythymidine) primers (Qiagen) and Super- Script II reverse transcriptase (Invitrogen). Quantitative RT-PCRs using the TaqMan mastermix (Applied Biosys- tems) were performed on a 7900HT Thermocycler (Applied Biosystems, Warrington, UK) using primers for pS2, PR and GAPDH cDNAs, purchased from Applied Biosystems. Firefly luciferase assay MELN cells (0.5 106) were plated in 24-well plates in medium containing DMEM/10% DSS for 24 h, trans- fected with CK1d siRNA for 48 h and treated with E2 (10 nM) or vehicle (ethanol) for 24 h. The cells were washed twice with PBS and lysed in 50 ml/well luciferase cell culture lysis reagent (Promega, Southampton, UK). Luciferase assays were performed using the ﬁreﬂy lucifer- ase assay system from Promega, according to the manu- facturer’s instructions, and measured with a Top Count NXT luminometer (Packard Biosciences, Beaconsﬁeld, UK). All experiments were performed independently at least three times, and results presented as the mean with standard error of the mean error bars. Data are normal- ized to the untreated sample, which was given a reference value of one. Western blotting and SDS–PAGE Whole cell lysates were prepared in NP40 lysis buﬀer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 10% (v/v) gly- cerol, 1% NP40, 5 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 50 mM leupeptin and 30 mg/ml apro- tinin). For western blotting, extracts were clariﬁed by centrifugation at 15 000g for 20 min at 48C and the pro- tein concentration of the lysates was determined using the bicinchoninic acid (BCA) protein assay (Pierce, Cramlington, UK). Lysates were then boiled in 5 sodium dodecyl sulfate (SDS) sample buﬀer (5 min, 958C), subjected to 12.5% SDS–PAGE and blotted on a Hybond C super nitrocellulose membrane (GE Healthcare). Following this, the membranes were blocked in TBS containing 0.1% (v/v) Tween20 and 5% (w/v) non- fat milk for 1 h, before probed overnight (O/N) with dif- ferent antibodies in the same buﬀer, and washed exten- sively in TBS/Tween. Immunocomplexes were detected by incubation for 45 min with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:1000 dilution), followed by enhanced chemiluminescence detection (ECL) (GE Healthcare). The intensity of bands were quantiﬁed using Image J software (NIH, Bethesda, MD). Immunoprecipitation Cells lysates were cleared by centrifugation (15 000 rpm, 10 min, 48C). Lysates containing equal amounts of pro- teins were precleared with IgG bound to protein A or G-agarose beads (Sigma) for 12 h at 48C and immunopre- cipitated with the speciﬁc primary antibody and protein A or protein G-agarose overnight with gentle agitation. The precipitates were then subjected to SDS–PAGE and immunoblotting using phosphospeciﬁc primary antibodies and horseradish peroxidase-labeled secondary antibodies. In vitro kinase assays In vitro kinase assays were carried out as described pre- viously (23). ERa-substrates that were used were: (i) full- length recombinant human ERa, (ii) GST-recombinant human ERa fragment encompassing the AF1 transactiva- tion domain and the DNA-binding domain (aa 1–280) and (iii) GST-recombinant human ERa fragment encompass- ing the ligand-binding domain (LBD) (aa 283–595). AIB1-substrates were: (i) full-length puriﬁed AIB1 and (ii) diﬀerent GST-recombinant human AIB1 fragments encompassing the RID region, responsible for ligand- dependent interaction with NRs (aa 582–800) (wt and those containing mutations at the following AIB1 aas: S601A, S664A, T714A, S715A and S794A). As a source of enzyme activity we used GST-tagged recombinant human CK1d protein (Invitrogen). Phosphorylated pro- teins were resolved by SDS–PAGE and the protein bands were visualized by autoradiography. Where indi- cated, the phosphorylated protein bands were excised and quantiﬁed by Cherenkov counting using LS-6500 scintillation counter (Beckman Coulter, San Francisco, CA, USA). Immunofluorescence MCF-7 cells were grown on poly-D-lysine-coated glass coverslips for 24 h in DMEM/10% DSS. Next, cells were transfected with CK1d siRNA or vehicle (ethanol) for 48 h and treated with E2 (10 nM) for 24 h. Cells were then washed twice in PBS and ﬁxed in methanol for 15 min at 208C. Fixed cells were washed with PBS and blocked with 0.2% gelatin in PBS for 1 h, before incubating them with AIB1 anti-rabbit antibody (1:300 in PBS) for 45 min at room temperature. After washing with PBS, coverslips were incubated for 45 min at RT with Alexa 488 secondary antibody (Invitrogen). DNA was visualized by DAPI staining. Cells were examined on an Axiovert-200 laser scanning inverted microscope (Zeiss, Welwyn Garden City, UK) equipped with a confocal imaging system. Statistical analysis Exploratory data analysis demonstrated that the distribu- tions were often skewed with outliers. Shapiro–Wilks test was used to test for normality (data were not normally distributed) and between group comparisons were made using the non parametric Mann–Whitney U-test.