Western blot chemiluminescence hrp substrate

For this assay, 100 μM of actin (Sigma-Aldrich Japan) and 50 μM of tubulin were denatured in unfolding buffer (6 M guanidine hydrochloride, 50 mM Tris-HCl pH 8.0, 100 mM KCl, 5 mM MgCl₂) at room temperature for 1 hour. The unfolded proteins were diluted into binding buffer (50 mM Tris-HCl pH 8.0, 100 mM KCl, 5 mM MgCl₂) and immediately applied to the StrepTrapHP column (GE Healthcare), which was bound with CtCCT^WT in advance. The bound proteins were eluted by binding buffer with 2.5 mM D-desthiobiotin. Proteins in the elution mixture were precipitated by the addition of trichloroacetic acid and then separated on 10% SDS gels. The gels were stained with Coomassie Brilliant Blue R-250. Transfer was conducted in transfer buffer (25 mM Tris, 200 mM glycine, 5% methanol) onto a 0.22 μm polyvinylidene difluoride (PVDF) membrane (Millipore). The primary antibody used for actin was the anti-β actin antibody (mAbcam 8224) from Abcam Co. Ltd. (Tokyo, Japan), and the antibody for tubulin was the monoclonal anti-α-tubulin antibody (T5168) from Sigma-Aldrich Japan (Tokyo, Japan). The secondary antibody was an anti-mouse IgG horseradish-peroxidase-linked whole antibody (NA931V, GE Healthcare). The membranes were visualized using the Western BLoT Chemiluminescence HRP Substrate (TaKaRa) and scanned with a Typhoon 8600 (GE Healthcare).

Whole or nuclear proteins (10 μg) were separated through electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel and then transferred onto an Immobilon-P membrane (Merck Millipore, Burlington, MA, USA). After blocking with 5% bovine serum albumin, each membrane was probed with primary antibodies for 1 h (Table 2). Secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were applied at a 1:20,000 dilution for 30 min. The membrane was incubated with Western BLoT Chemiluminescence HRP Substrate (TaKaRa Bio), and membrane image was captured using a chemiluminescent imaging system LuminoGraph I (ATTO, Tokyo, Japan). The density of each protein band was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Yin Yang 1 (YY1) were used as loading controls for whole and nuclear proteins, respectively. The phosphorylation levels of the target proteins were calculated as the ratio of their values to those of the corresponding total protein as a loading control.

Immunoprecipitation complexes obtained from ChIP-TH2B/ ChIP-IgG were eluted in 2X Laemmli buffer and electrophoresed on 15% SDS-PAGE at 100 V for 2.5 h. Proteins were transblotted on Nitrocellulose membrane (Pall bioscience, Pensacola, FL, USA) at 100 V for 1 h 15 min. Non-specific binding to the membrane was blocked with 5% NFDM incubated on a rocker for 1 h at RT. After blocking, the membrane was incubated with anti-TH2B antibody diluted 1:5000 in 1% NFDM and kept overnight at 4 °C with constant rocking. The following day, blots were washed thrice with 0.1% PBST for 5 min and incubated with 1:3000 diluted Protein-G HRP (Thermo fisher scientific, Waltham, MA, USA), which served as secondary antibody, for 45 min with constant rocking. The blots were washed thrice with 0.1%PBST for 5 min and developed using Western Blot Chemiluminescence HRP substrate (TAKARA BIO INC, Otsu, Shiga, Japan).