Tris hcl

All staple DNAs used to prepare the DNA hexagonal unit were purchased from Operon Biotechnology (Tokyo, Japan). Single-stranded M13mp18 viral DNA was purchased from New England Biolabs, Inc (Ipswich, MA). The gel-filtration column and the Sephacryl S-300 were purchased from BioRad Laboratories (Hercules, CA) and GE Healthcare (Buckinghamshire, UK), respectively. Tris-HCl, EDTA and MgCl₂ for electrophoresis analysis were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Water was deionized (18.0 MΩ cm specific resistance) by a Milli-Q system (Millipore Corp., Bedford, MA). Streptavidin was purchased from Wako Pure Chemical Industries (Osaka, Japan). DOPC was obtained from Avanti Polar Lipids (Alabaster, AL) as chloroform stocks.

The upstream sequence of ZFTA from − 3149 to +2 (corresponding to the second nucleotide of ZFTA’s first codon) was amplified by PCR using a human bacterial artificial chromosome clone (RP11-179P19, Advanced GenoTechs), which lacks the − 154th guanine base, as a template, and cloned into a pGL4.26 (luc2/minP/Hygro) vector (Promega). A series of deletion mutants for this plasmid was generated by PCR using PrimeSTAR MAX or Tks Gflex DNA polymerase (TaKaRa). HeLa cells were transfected with the indicated plasmids listed in Table S1 using HilyMax (Dojindo) and incubated overnight. Luciferase assay was performed using TriStar LB 941 (Berthold) and the following reaction buffers: 100 mM Tris–HCl (pH7.8, Nacalai Tesque), 5 mM MgCl₂ (Wako Pure Chemical), 250 µM Coenzyme A trilithium salt (Oriental Yeast), 150 µM adenosine triphosphate (Nacalai Tesque), 150 µg/mL D-Luciferin potassium salt (Cayman Chemical), 0.5 mM dithiothreitol (Nacalai Tesque), 50 µM ethylenediaminetetraacetic acid (pH8.0, Dojindo) and 0.1% Triton X-100 (Sigma-Aldrich).

IP was performed with Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s protocol. The preincubation of Dynabeads (100 µl) (M-280 Sheep Anti-Mouse IgG or M-280 Sheep Anti-Rabbit IgG) with primary antibody (5.0 µl) in 1.0% BSA/PBS was performed at 4°C overnight. The beads conjugated with primary antibodies were then washed with IP buffer (10 mM Tris–HCl [pH 7.8] [Nacalai Tesque], 1.0% NP-40 [Nacalai Tesque], and 15 mM NaCl [Nacalai Tesque]/EDTA-free protease inhibitors [100×] [Nacalai Tesque]). Cells were collected with a cell scraper in IP buffer. A total of 1.0 × 10⁷ cells were sonicated on ice three times for 1 s by the XL-2000 sonicator (MISONIX). Sonicated cells were centrifuged at 21,900g for 10 min. The supernatant was collected and incubated with primary antibody conjugated with Dynabeads at 4°C overnight. After incubation, the sample tubes were set on DynaMag-2 (Thermo Fisher Scientific), and Dynabeads were washed three times with IP buffer. Dynabeads were then suspended with fresh IP buffer and incubated at 95°C for 5 min. The supernatant was collected on DynaMag-2 and mixed with 2 × Laemmli sample buffer. Immunoblotting was performed as described.