Pcdna3.1 his

The EtLPP plasmids were generated by insertion of the E. tenella LPPs sequence into the mammalian expression vector pcDNA3.1/His (Invitrogen, Grand Island, NY, USA). HEK 293 cells were plated into six-well dishes and grown to 50–60% confluence in 10% CO₂. Transfection was performed using Lipofectamine, OPTI-MEM medium (Life Technologies, Gaithersburg, MD, USA), and 1–5μg of recombinant pcDNA3.1/His-expressing tagged EtLPP DNA per well. Control cells were transfected with the corresponding pcDNA3.1/His-vector DNAs. The DNA-containing medium was removed from the plate after 24h, and replaced by DMEM containing 10% FBS. After 48h, cells were washed and sonicated in lysis buffer [29 (link)]. The membrane fractions were prepared according to the method described previously [29 (link)], though with some modification (Ni-NTA agarose resin (Qiagen, Hilden, Germany) was used for protein immunoprecipitation). Protein concentration was determined by Bradford protein assay. SDS-PAGE and immunoblot analysis were conducted according to existing protocols [29 (link)].

Full-length BCAS2, as obtained in the yeast two-hybrid screen, was amplified from pACTII-BCAS2 and cloned into pcDNA3.1/His (Invitrogen, Carlsbad, CA, USA) to make pcDNA-BCAS2. All glutathione-S-transferase (GST) fusion constructs were generated in a pDEST15 vector, as previously described [42 (link)]. Estrogen and progesterone receptor vectors (pSG5-ERα and pSG5-PR) were donated by Dr. Pierre Chambon (INSERM, France). ER activation domain expression vectors, pERα-AF1 (aa 1-283) and pERα-AF2 (aa 144-595), as well as 3X-ERE-Luc were provided by Dr. Donald McDonnell (Duke University, USA) [17 (link)]. pSG5-SRC-1, pSG5-CBP and pSG5-TIF2 were obtained from Dr. Edwin Milgrom (INSERM, France).

The assay for NHEJ was performed as previously described [3] (link). DNA substrate with either complementary or blunt ends was prepared by linearizing pcDNA3.1-His (Invitrogen, USA) with EcoRI or EcoRV. DNA fragments (5.5 kb) were purified using Tiangen spin columns and resuspended to a concentration of 5 µg/ml. End joining reactions (20 µl) were performed with 60 µg of protein extract and 40 ng of DNA substrate in the presence of T4 ligase buffer at 37°C for 2 h. Samples were incubated with RNase A (80 µg/ml) at 37°C for 10 min. The protein was then removed by incubation with proteinase K (2 mg/ml) and 0.5% (w/v) SDS at 37°C for 10 min and extracted using Tris-buffered phenol/chloroform/isoamyl alcohol. DNA separation was performed by agarose (0.7%) gel electrophoresis with SYBR Gold (Invitrogen, USA) staining as described above. Imaging was conducted using Gel-Pro Analyzer software ver.6.0 (Media Cyberetics, USA). The intensities of the DNA bands were quantified using computerized gel image analysis software (Gel-Pro Analyzer software ver.6.0, Media Cyberetics, USA).