Figure EV2. Neddylation of FLAG‐NSUN2 is not readily detected in WT or NEDP1 KO cells.

WT and NEDP1 KO HEK 293 cells were transfected with FLAG‐NSUN2 and 48 h later harvested for anti‐FLAG immunoprecipitation. Following stringent washes, pulldowns were treated with recombinant GST‐NEDP1, the deubiquitinase his₆‐USP1/UAF1, or left untreated. After incubation at 30°C for 30 min, reactions were stopped by the addition of LDS sample buffer, and then, samples were processed for Western blot analysis with the indicated antibodies. There is no shift in the anti‐FLAG immunoreactive band, which suggests NSUN2 is neither modified by NEDD8 or ubiquitin.