4.8. Ubiquitination and Deubiquitination Assays

For ubiquitination assay, Myc-Bax, and HA-Ub were co-transfected into HeLa cells. The cell lysates were used for immunoprecipitation with an anti-Myc antibody. They were incubated with an antibody at 4 °C overnight and then for 2 hrs with protein A/G Plus-Agarose Beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western blotting was performed with the cell lysates. For deubiquitination assay, Flag-USP12, Myc-Bax, and HA-Ub were co-transfected into HeLa cells. Immunoprecipitation was performed with an anti-Myc antibody. Both assays were performed using a ubiquitination assay kit, according to the manufacturer’s manual (cat. no. UBAK-100; D&P Biotech Inc. Seoul, Korea).