Cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich; EMD Millipore) as previously described (22 (link)–24 (link)). Cells were centrifuged at 4°C for 10 min at 16,000 × g. Protein concentrations were determined by the Bradford assay (25 (link)). Aliquots containing 20 µg of total protein were separated by 10% sodium dodecyl-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (1:5,000, mouse monoclonal; A5316; Sigma-Aldrich; EMD Millipore). Appropriate secondary antibodies (1:3,000; rabbit anti-goat; ab6741; 1:5,000; rabbit anti-mouse; ab97046; Abcam, Cambridge, MA, USA) conjugated to horseradish peroxidase and enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) were used to detect the bound primary antibodies. Co-IP was performed with cell lysate (500 µg) incubated with USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc.), relA (1:1,000; rabbit polyclonal; sc-372; Santa Cruz Biotechnology, Inc.) or non-specific-IgG antibodies (1:1,000; rabbit IgG, monoclonal; ab172730; Abcam) using µMACS™ Protein A/G MicroBeads and MACS® Separation Columns according to the manufacturer's protocol (Miltenyi Biotec, Auburn, USA).
Peng L., Hu Y., Chen D., Linghu R., Wang Y., Kou X., Yang J, & Jiao S. (2016). Ubiquitin specific protease 21 upregulation in breast cancer promotes cell tumorigenic capability and is associated with the NOD-like receptor signaling pathway. Oncology Letters, 12(6), 4531-4537.
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