Restriction and dna modifying enzymes

The growth and maintenance of E2 responsive and ERα-synthesizing MCF7 and T47D cells were described previously^{4 (link),64 (link)}. In all experiments, media were changed every third day when appropriate.
Restriction and DNA modifying enzymes were obtained from New England Bio-Labs (Beverly, MA, USA) or Thermo-Fischer Sci. (Waltham, MA, USA). 17β-estradiol (E2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The complete antagonist of estrogen receptor, Imperial Chemical Industries 182780, (ICI) was obtained from Tocris Biosciences (Ellisville, IL, USA). The antibody for Poly(ADP-ribose) polymerase 1 (PARP1; 9542) was obtained from Cell Signaling Technology (Beverly, MA, USA). The antibodies for CXXC5 (ab106533) and for HDAC1 (ab19845) were purchased from Abcam Inc. (Cambridge, MA, USA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). siRNAs (FlexiTube siRNA) for CXXC5 were purchased from Qiagen Inc. (Düsseldorf, Germany). Camptothecin (13637) was purchased from Cell Signaling Technology.

[γ-³²P]ATP, [¹⁴C]leucine, and [³H]leucine were from Perkin-Elmer (Waltham, MA, USA). All restriction and DNA-modifying enzymes were from New England Biolabs (Ipswich, MA, USA). Rabbit reticulocyte lysate was from Ambion (Austin, TX, USA), and SP6 RNA polymerase was prepared as described.^{23 (link)}

CHO (Hprt^−/−) cells (JCRB0218) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). CHO K1 cells (RCB0285) and DT40 cells (RCB1464) were obtained from the Riken BioResource Research Center (Ibaraki, Japan). P3X63Ag8.653 myeloma cells (CRL-1580) and HCT116 cells (CCL-247) were purchased from the American Type Culture Collection (Manassas, VA, USA). TT2F cells were provided by RIKEN Centre for Biosystems Dynamics Research (Hyogo, Japan). Restriction and DNA-modifying enzymes were purchased from New England Biolabs (Ipswich, MA, USA) and TOYOBO (Tokyo, Japan), respectively. Primers were obtained from Eurofins (Huntsville, AL, USA). E. coli strains DH5α and Rosseta-gami B(DE3) pLysS were purchased from Takara Bio (Shiga, Japan) and Merck Millipore (Burlington, MA, USA), respectively.

DNA restriction and modifying enzymes were from New England Biolabs and Roche Applied Science; T4 DNA ligase was from Promega; anti-GFP from Roche; anti-RyR (34C, which recognizes both RyR1 and RyR2, DSHB) from the Developmental Studies Hybridoma Bank). The antibody against a junctin peptide (SKHTHSAKGNNQKRKN, with a cysteine added to the N-terminus and conjugated to KLH, GL Biochem, Shanghai) was from IMVS Veterinary Services, Australia. PCR primers were from GeneWorks, Australia.

PCR products were prepared using proofreading Phusion DNA polymerase (Finnzymes) and oligonucleotide primers from Integrated DNA Technologies, unless otherwise stated. DNA restriction and modifying enzymes were from New England Biolabs. PCR products were purified and gel-extracted using kits from either Qiagen or Geneaid. DNA sequences of all constructs were verified using an Applied Biosystems Model 3100 DNA sequencer. E. coli strain DH5 alpha was used as host for molecular cloning. Yeast strains S. cerevisiae YVH10 or P. pastoris SuperMan5 (Research Corporation Technologies) were used as hosts for soluble protein secretion and S. cerevisiae EBY100 for cell surface display.

All recombinant DNA techniques were performed according to standard procedures using E. coli DH5α as the initial host. DNA restriction and modifying enzymes were obtained from New England Biolabs and used according to the manufacturer’s recommendations. Primers used are shown in Table S1.