Horseradish peroxidase labeled anti rabbit igg antibody

Cell lysates were prepared using NP-40 lysis buffer (0.5% Non-idet P-40, 5 mM EDTA, 2 mM Na₃VO₄, 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mg/mL aprotinin, 1 mM PMSF). Protein concentration was determined using the Protein Assay Dye (Bio-Rad Laboratories, Hercules, CA, United States). Proteins (20 μg) were separated using 12% SDS-PAGE and transferred to a PVDF membrane (GE Healthcare). The membrane was probed with anti-β-catenin antibody (#8480; Cell Signaling, Danvers, MA, United States) or anti-β-actin antibody (013-24553; Fujifilm Wako Pure Chemicals, Osaka, Japan). Horseradish peroxidase-labeled anti-rabbit IgG antibody (GE Healthcare) and anti-mouse IgG antibody (GE Healthcare) were used as the secondary antibodies. The proteins were detected using an ImmunoStar LD chemiluminescence detection kit (Fujifilm Wako Pure Chemicals) and visualized with a LAS-1000 Lumino Image analyzer (Fujifilm, Tokyo, Japan).

U937 and HL-60 cells were purchased from ATCC (Manassas, VA, United States). AZA-resistant cell lines (R-U937 and R-HL-60) were originally created in our laboratory from U937 and HL-60 cells, respectively (Imanishi et al., 2014 (link)).
5-Azacytidine was purchased from Wako Pure Chemical Industries (Osaka, Japan) and I-BET151 from Selleck Chemicals (Houston, TX, United States). The antibodies specific for HP1α, HP1β, HP1γ, and β-actin were purchased from Abcam (Cambridge, United Kingdom). The antibodies specific for BRCA1 phosphorylated at Ser 1423 (p-BRCA1), total BRCA1 and total ATM were from Santa Cruz Biotechnology (Dallas, TX, United States) and anti ATM phosphorylated at Ser 1981 (p-ATM) antibody was from R&D Systems (Minneapolis, MN, United States). Anti PARP1 antibody was from Cell Signaling Technologies (Danvers, MA, United States). The secondary antibodies, namely horseradish peroxidase–labeled anti-mouse IgG antibody and horseradish peroxidase–labeled anti-rabbit IgG antibody, were purchased from GE Healthcare (Buckinghamshire, United Kingdom).

Cell lysates were resolved by electrophoresis using 12% SDS-PAGE and transferred to a Hybond P membrane (GE Healthcare). The membrane was incubated with anti-SMARCD1 antibody (10998-2-AP; Proteintech, Rosemont, IL) or anti-β-actin (013-24553; Applied Biological Materials Inc., Richmond, BC, Canada). Horseradish peroxidase-labeled anti-rabbit IgG antibody (GE Healthcare) and ant-mouse IgG antibody (GE Healthcare) was used as secondary antibodies. The proteins were detected using ImmunoStar LD (Wako Pure Chemical, Osaka, Japan) and visualized with a LAS-1000 Lumino Image analyzer (Fuji Film, Tokyo, Japan).