Las 4000 image analyzer

The purified nucleosome (0.38 μM), containing H3.1 or CENP-A, was incubated with trypsin (2.5, 5.0, 7.5, 10 ng, purchased from Sigma-Aldrich) in 10 µl of reaction solution, containing 8 mM Tris-HCl (pH 7.5), 0.5 mM MgCl₂, and 1 mM DTT, at 25 °C for 1 min. The reaction was stopped by adding 10 µl of a 4% SDS solution, containing 0.10 M Tris-HCl (pH 6.8), 20% glycerol, and 0.2% bromophenol blue, and the samples were boiled at 95 °C for 15 min. For the western blotting, 6.0 µl portions of the samples were analyzed by 18% SDS-PAGE at 200 V for 100 min. After SDS-PAGE, the western blotting analyses were performed by the method described above, using an anti-H4 rabbit polyclonal antibody (1:1000; Abcam, ab7311) and an HRP-anti rabbit-IgG F(ab’)₂ fragment (1:5000; GE Healthcare, NA9340) as the primary and secondary antibodies, respectively. The blocking, primary antibody reaction, and secondary antibody reaction were performed at 4 °C overnight, 4 °C for 3–12 h, and 4 °C for 1–2 h, respectively. The membrane was washed with PBS-T, and then treated with Amersham ECL Prime (GE Healthcare). Chemiluminescence was detected with an LAS4000 image analyzer (GE Healthcare). The uncropped images of all blots are presented in Supplementary information. The uncropped images of all blots are presented in Supplementary Figure 11.

Strains were cultured on DPY medium for 18 h. The fungal mycelia were frozen in liquid nitrogen and subsequently ground using a multi-bead shocker. After extraction, cell lysates were incubated with a buffer containing detergent NP-40 [50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM/mL PMSF, 1× Protease Inhibitor Cocktail (Sigma, St. Louis, MO, USA: P8215), 1% NP-40] to solubilize the membrane proteins. Mycelial extract solubilized with the buffer was incubated in ice for 20 min. After centrifugation at 1000×g for 10 min, the supernatant was collected. Cell lysates were separated using SDS-PAGE. The proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Burlington, MA, USA) using a semidry blotting system (Nihon Eido, Tokyo, Japan). To detect EGFP, Living Colors® A.V. (anti-GFP) monoclonal antibody (1: 2000 dilution, cat # 632380, Clontech) and peroxidase-labeled anti-mouse IgG (H + L) antibody (1: 2000 dilution, cat # PI-2000, Vector Laboratories, Newark, CA, USA) were used as primary and secondary antibodies, respectively. Chemiluminescence was detected using a Western Lightning-ECL system (PerkinElmer, Waltham, MA, USA) and an LAS-4000 image analyzer (GE Healthcare, Buckinghamshire, UK).

Whole-cell lysates, prepared with SDS sample buffer containing protease (Nakalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktails (Calbiochem), were separated by SDS-PAGE and were transferred to PVDF membranes (Immobilon-P; Millipore). The membranes were incubated at room temperature with primary antibodies for 2 h and then with HRP-conjugated secondary antibodies for 1 h. The membranes were treated with ECL reagent (GE Healthcare, Buckinghamshire, UK) and signals were detected using an LAS-4000 Image Analyzer (GE Healthcare). ImageQuant TL software (GE Healthcare) was used to quantitate the intensity of bands. The Ready-to-Use Mouse Mixed Tissue Western Blot (Panel 5; Zyagen Laboratories, San Diego, CA, USA) PVDF membrane was used to analyze BMCC1 expression in mouse tissues.

For preparation of nuclear extract, the PBMCs from two unrelated healthy blood donors were isolated using lymphocyte separation solution (Ficoll, GE, USA). Then, nuclear extracts were derived from PBMCs using the Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo, USA) according to the manufacturer's instructions. Oligonucleotides (29 base pairs) were designed corresponding to genomic sequences surrounding SNP rs1491941. Single-stranded oligonucleotide probes were labeled using the DIG Gel Shift Kit (Roach, Germany), and sense and antisense oligonucleotides were then annealed. DNA-protein interactions were detected by using a DIG Gel Shift Kit (Roach, Germany) according to the manufacturer's instructions. EGR 1 antibody (ab55160, abcam, USA) was used for supershift analysis. The DNA-protein complexes were separated on a non-denaturing 5% polyacrylamide gel in 0.5 × Tris-borate-EDTA (TBE) running buffer for 120 min at 50 V. The gel was transferred to a nitrocellulose membrane, and signals were detected using a LAS-4000 Image analyzer (GE, USA).

The total protein was extracted from log-phase cells with an NuPAGE LDS sample buffer (ThermoFisher) after 0.2N NaOH treatment (Kushnirov, 2000 (link)). For each analysis, the total protein extracted from two optical density (OD) units of cells with OD₆₀₀ was used. For total protein visualization, the extracted total protein was labeled with Ezlabel FluoroNeo (ATTO), as described in the manufacturer’s protocol, and separated by 4–12% SDS-PAGE. Proteins were detected and measured using the LAS-4000 image analyzer (GE Healthcare) in SYBR–green fluorescence detection mode and Image Quant TL software (GE Healthcare). The expression of each target protein (AU) was calculated, as shown in Figure 2. Average values, SD, and p-values of Welch's t-test were calculated from biological triplicates. For detection of Tpi1, the SDS-PAGE-separated proteins were transferred to a PVDF membrane (ThermoFisher). Tpi1 was detected using an anti-Tpi1 antibody (RRID:AB_11130951), a peroxidase-conjugated secondary antibody (Nichirei Biosciences), and a chemiluminescent reagent (ThermoFisher). The chemiluminescent image was acquired with an LAS-4000 image analyzer in chemiluminescence detection mode.