Figure 6. Cytosolic Nmnat2 mutants exhibit increased protein stability and reduced levels of ubiquitination.

(A) Representative Western blots of HEK293 cells co-transfected with FLAG-Wld^S and the indicated FLAG-Nmnat2 variant. Twenty-four hours after transfection, cells were treated with 10 µM emetine for the amount of time indicated after which samples were processed for SDS-PAGE and Western blot using anti-FLAG antibody. (B) Quantification of Nmnat2 turnover after emetine treatment. For each sample and time point, the amount of FLAG-Nmnat2 remaining was normalised to FLAG-Wld^S as an internal control. Error bars indicate SEM. (C) Representative Western blots of GST-Dsk2 pulldown assay. HEK293 cells expressing a FLAG-Nmnat2 variant were lysed (inp. – total input) and ubiquitinated proteins were immunoprecipitated using GST-Dsk2 bound to glutathione beads (ubiq.). GST-fused mutant Dsk2 was used for control pulldown (cont.). Eluted proteins were processed for SDS-PAGE and analysed by Western blot using anti-FLAG antibody. (D) Quantification of ubiquitination assay. For each Nmnat2 variant, the total amount of ubiquitinated FLAG-Nmnat2 was normalised to total input. Error bars indicate SEM. ** and *** indicate statistically significant difference compared to wild-type Nmnat2 (** p<0.01*** p<0.001).