U2OS cells transfected with 3 µg of plasmids pEGFP-C1, pEGFP-WBSCR22, pEGFP-N6AMT1iso1, pEGFP-N6AMTiso2 and treated with 5 µM MG132 were used for co-immunoprecipitation with GFP-Trap_M magnetic beads (ChromoTek GmbH, Munich, Germany) following the manufacturer’s protocol. Alternatively, co-immunoprecipitation was carried out with HeLa cells transfected with 3 µg of pEGFP-C1, pEGFP-WBSCR22, pEGFP-N6AMT1iso1, pEGFP-N6AMT1-R111D117/AA, pEGFP-N6AMTiso2 and treated with 0 µM or 5 µM MG132.
The proteins were detected by western blot using the mouse monoclonal antibodies anti-E2-Tag antibody 5E11 (Icosagen), anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA) and rabbit polyclonal antibodies against TRMT112 (HPA04006, Sigma-Aldrich), WBSCR22 (sc-135322; Santa Cruz Biotechnology, Dallas, TX, USA), EGFP (Institute of Technology; University of Tartu, Tartu, Estonia) and N6AMT1 (ab173804; Abcam, Cambridge, UK). Detection was performed using an ECL detection kit (GE Healthcare, Chicago, IL, USA) following the manufacturer’s instructions.
The proteins were detected by western blot using the mouse monoclonal antibodies anti-E2-Tag antibody 5E11 (Icosagen), anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA) and rabbit polyclonal antibodies against TRMT112 (HPA04006, Sigma-Aldrich), WBSCR22 (sc-135322; Santa Cruz Biotechnology, Dallas, TX, USA), EGFP (Institute of Technology; University of Tartu, Tartu, Estonia) and N6AMT1 (ab173804; Abcam, Cambridge, UK). Detection was performed using an ECL detection kit (GE Healthcare, Chicago, IL, USA) following the manufacturer’s instructions.