4.10. SUMOylation Assay

HeLa cells (2 × 10⁶) were co-transfected with the expression constructs in 10 cm dishes with 2 μg each of His-SUMO1 and HA-tagged wild-type HMGXB4 or mutants. At 48 h post-transfection, cells were lysed by radioimmunoprecipitation assay (RIPA) buffer containing 10 mM N-ethylmaleimide (NEM) and protease inhibitors. For immunoprecipitation, equal amounts of lysates (containing 5 mg of total cellular protein from HeLa cells) were precleared with protein G-agarose beads (Sigma, St. Louis, MO, USA). Precleared extracts were incubated with 1 μg rat monoclonal anti-HA (Roche, Mannheim, Germany) for 2 h at 4 °C. Precipitates were washed extensively in lysis buffer; bound complexes were eluted with 2 × SDS–PAGE sample buffer and resolved by 7.5–10% SDS–PAGE. Immunoblotting was performed according to standard procedures, and proteins were detected with the anti-HA antibodies. Antibodies were detected by chemiluminescence using ECL Advance Western Blotting Detection Kit (GE Healthcare, Chicago, IL, USA).