Ubiquitination Analysis of MFF and TRAF3

For ubiquitination analysis, 293T cells were co-transfected with LPC-HA-Ub and pUB-TRAF3-SBP-6xHis, pUB-Myc-MFF or an empty lentiviral vector pUB-Thy1.1. At day 2 post transfection, cells were treated with 10 µM of the proteasome inhibitor MG-132 at 37°C for 4 h, and then harvested at 2 x 10⁷ cells/condition. Total cellular proteins were lysed in 1% CHAPS Lysis Buffer (39 (link)) containing 1x Phosphatase Inhibitors (Pierce) and 1 mM NEM. The insoluble pellets were removed by centrifugation at 10,000 g for 20 minutes at 4°C. The CHAPS lysates were subsequently immunoprecipitated with the Anti-c-Myc Tag (9E10) Affinity Gel (BioLegend) or Streptavidin-Sepharose beads (Pierce). Immunoprecipitates were washed 5 times with the Wash Buffer (39 (link)) containing 1x Phosphatase Inhibitors and 1 mM NEM. Ubiquitination of the immunoprecipitated MFF or TRAF3 was analyzed by immunoblot analyses.