Western Blot Analysis of Protein Interactions

Protein extracts were separated on 7.5 or 10% Mini-PROTEIN TGX Precast Gels (Bio-Rad) and then transferred onto nitrocellulose membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). The following primary antibodies and dilutions were used: rabbit anti-PRDM9 polyclonal (Grey et al. 2017 (link)), 1:500; rabbit anti-CTCF monoclonal (Cell Signaling Technology, D31H2), 1:2000; rabbit anti-CXXC1 polyclonal (Millipore, ABE211), 1:5000; rat anti-tubulin α monoclonal (Abcam, ab6161), 1:3000; rabbit anti-PIH1D1 polyclonal (Proteintech, AP19427), 1:5000; rabbit anti-CEP70 polyclonal (Abnova, PAB17658), 1:1000; mouse anti-MCRS1 polyclonal (Abnova, H00010445-B01P), 1:250; anti-Gal4 AD (Millipore, 06-283), 1:3000; and anti-Gal4 BD (Sigma, G3042), 1:2000. Signals were detected with the horseradish peroxidase (HRP)-conjugated secondary antibodies, donkey anti-rabbit IgG HRP (Jackson ImmunoResearch Laboratories), goat anti-rat IgG HRP (GE Healthcare), and sheep anti-mouse IgG HRP (GE Healthcare), and then visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). ImageJ 1.49m was used to quantify signal intensities of western blot images captured by ChemiDoc™ XRS+ Imager with the Image Lab 5.1 software.