The SCFFBXO21-mediated ubiquitination at K155 within the ISTID underscores the unique lability of NMNAT2. (A) A schematic of in vitro ubiquitination of NMNAT2 by the SCFFBXO21 E3 ligase and detection of the ubiquitination sites using the LC-MS/MS. (B) The major domains of NMNAT2 and the ubiquitination sites detected in MS are shown. (C) Representative image of the denaturing IP and western blot analysis of the ubiquitination levels of WT and mutant NMNAT2-FLAG proteins in N2a cells as indicated. The experiments were repeated four times. (D) Representative image of western blot analysis examining the ubiquitination levels of purified WT or K155R mutant -NMNAT2 protein after the in vitro ubiquitination by SCFFBXO21. The experiments were repeated three times. (E and F) The representative image (E) and quantification (F) of the western blot analysis of the turnover of WT or K155R NMNAT2-FLAG protein in N2a cells following the CHX treatment. All protein levels are normalized to GAPDH, and the relative level at 0 h is set to 100%. (G) K155 and the flanking residues of NMNAT2 are conserved across different species of mammals. (H) The evolutionary relationship of the three NMNAT proteins in different species of vertebrates (see Materials and methods). (I) Sequence alignment of the three human NMNAT proteins (see Materials and methods). The red box highlights the highly variable ISTIDs. (J) Schematic diagrams of the swapping of the ISTID domain of NMNAT2 with that of NMNAT1 or NMNAT3 and the K155R mutation. (K–N) Representative images (K and L) and quantifications (M and N) of the CHX chase assay to assess the protein turnover rate of NMNAT1-FLAG (K and M) or NMNAT3-FLAG (L and N) as indicated in N2a cells. All protein levels are normalized to GAPDH, and the relative level at 0 h is set to 100%. Mean ± SEM. n = 6 in F and n = 3 in M and N. Two-way ANOVA; *P < 0.05 and ***P < 0.001; ns, not significant. Source data are available for this figure: SourceData F7.
Figure 7.

The SCF FBXO21 -mediated ubiquitination at K155 within the ISTID underscores the unique lability of NMNAT2. (A) A schematic of in vitro ubiquitination of NMNAT2 by the SCFFBXO21 E3 ligase and detection of the ubiquitination sites using the LC-MS/MS. (B) The major domains of NMNAT2 and the ubiquitination sites detected in MS are shown. (C) Representative image of the denaturing IP and western blot analysis of the ubiquitination levels of WT and mutant NMNAT2-FLAG proteins in N2a cells as indicated. The experiments were repeated four times. (D) Representative image of western blot analysis examining the ubiquitination levels of purified WT or K155R mutant -NMNAT2 protein after the in vitro ubiquitination by SCFFBXO21. The experiments were repeated three times. (E and F) The representative image (E) and quantification (F) of the western blot analysis of the turnover of WT or K155R NMNAT2-FLAG protein in N2a cells following the CHX treatment. All protein levels are normalized to GAPDH, and the relative level at 0 h is set to 100%. (G) K155 and the flanking residues of NMNAT2 are conserved across different species of mammals. (H) The evolutionary relationship of the three NMNAT proteins in different species of vertebrates (see Materials and methods). (I) Sequence alignment of the three human NMNAT proteins (see Materials and methods). The red box highlights the highly variable ISTIDs. (J) Schematic diagrams of the swapping of the ISTID domain of NMNAT2 with that of NMNAT1 or NMNAT3 and the K155R mutation. (K–N) Representative images (K and L) and quantifications (M and N) of the CHX chase assay to assess the protein turnover rate of NMNAT1-FLAG (K and M) or NMNAT3-FLAG (L and N) as indicated in N2a cells. All protein levels are normalized to GAPDH, and the relative level at 0 h is set to 100%. Mean ± SEM. n = 6 in F and n = 3 in M and N. Two-way ANOVA; *P < 0.05 and ***P < 0.001; ns, not significant. Source data are available for this figure: SourceData F7.

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