Investigating Protein Ubiquitination and Interactions

WB, in vitro and in vivo ubiquitination, and co-IP assays were carried out as previously described (18 (link), 33 (link), 41 (link)). The following primary antibodies were used for immunoblotting: anti-Flag (1:5000; Sigma-Aldrich, F7425), anti-Myc (1:5000; Proteintech, 16286-1-AP), anti-hemagglutinin (1:5000; Sigma-Aldrich, H3663), anti-RNF220 (1:1000; Sigma-Aldrich, HPA027578), anti-MBP (1:1000; Sigma-Aldrich, ab9348), anti-PDGFRα (1:1000; BD Biosciences, 558774), anti–glial fibrillary acidic protein (GFAP) (1:1000; Proteintech, 60190-1-Ig), anti-PLP (1:1000; Cell Signaling Technology, 85971S), anti-MOG (1:1000; Proteintech, 12690-1-AP), anti-MAG (1:1000; Proteintech, 14386-1-AP), anti-Olig1 (1:1000; Santa Cruz Biotechnology, SC-166257), anti-Olig2 (1:1000; Millipore, MABN50), anti-ubiquitin (1:1000; Santa Cruz Biotechnology, SC-8017), and anti–α-tubulin (1:5000; Proteintech, 66031-1-Ig). Horseradish peroxidase–coupled goat anti-mouse or rabbit antibodies (Pierce, 31430 and 31460) were used as secondary antibodies. Chemiluminescence detection was conducted using a chemiluminescent protein detection kit (Thermo Fisher Scientific, 34580) according to the manufacturer’s instructions. Immunoblot signals were quantified by ImageJ software.