Sc 365685

Tissue specimens were immunostained with an Ultra-Vision Detection System (Thermo Scientific) following the manufacturer’s protocol. Primary rabbit polyclonal antibodies against budding uninhibited by benzimidazoles 1 (BUB1; 1:50, sc-365685; Santa Cruz Biotechnology), cyclin-dependent kinase 1 (CDK1; 1:100, HPA003387; Sigma-Aldrich), maternal embryonic leucine zipper kinase (MELK; 1:200, HPA017214; Sigma-Aldrich), and non-SMC condensin I complex subunit G (NCAPG; 1:150, ab56382; Abcam) were used for immunochemistry. The procedures were described previously (Goto et al, 2015 (link); Goto et al, 2016a (link); Goto et al, 2016b (link)).

Protein lysates were extracted from fibroblasts. A total of 25 μg of proteins were loaded on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4561034; Bio-Rad, Hercules, CA) and transferred to a nitrocellulose membrane. Membranes were probed against SGO1 (mouse monoclonal, ab58023; Abcam, Cambridge, UK), BUB1 (mouse monoclonal, sc-365685; Santa Cruz, Dallas, TX), SM22α (rabbit polyclonal, sc-50446; Santa Cruz), and TPM1 (rabbit polyclonal, sc-28543; Santa Cruz) primary antibodies diluted 1:200 followed by horseradish-peroxidase–conjugated secondary antibodies. The blots were developed using SuperSignal (34580; Thermo Scientific). Signal intensity was quantified using ImageJ software (v.1.48; National Institutes of Health, Bethesda, MD).⁴⁵ (link)