Immunoprecipitation of Myc-UBL3 from MutuDC1940 cells

Ubl3KO MutuDC or Ubl3KO MutuDC stably expressing either Myc-UBL3(WT) or Myc-UBL3(C114S) were lysed in 1% Triton buffer on ice and nuclei removed by centrifugation at 14,000 × g at 4 °C. Postnuclear supernatants of 5 x 10^6 cells were incubated with anti-Myc-Tag (9B11) mouse mAb conjugated to magnetic beads (Cell Signaling Technologies) at 4 ºC, washed and eluted by acidification with 0.1% FA and denatured with 2,2,2-trifluoroethanol. Proteins were reduced with 12.5 mM tris(2-carboxyethyl)phosphine (TCEP), and digested with 0.25 mg proteomics grade Trypsin (Sigma-Aldrich) in 50 mM triethylammonium bicarbonate (TEAB)) overnight at 37 °C. Peptides were dried by SpeedVac, reconstituted in 2% acetonitrile and 0.05% trifluoroacetic acid, centrifuged for 10 minutes at 21,000 × g to remove particulate matter, and filtered over Vivacon 500 30kDa columns (Satrorius).