The SCFFBXO21E3 ligase complex promotes NMNAT2 ubiquitination in vivo and in vitro. (A and B) Representative image (A) and quantification (B) of the denaturing IP and western blot analysis of the ubiquitination levels of NMNAT2 in N2a cells with Fbxo21 KD. NMNAT2-FLAG is immunoprecipitated from denatured cell lysates with an anti-FLAG antibody, and the ubiquitinated NMNAT2 in the IPs is detected with an anti-HA antibody for (HA-Ub). Ub: ubiquitin. The relative levels of ubiquitinated NMNAT2 are normalized to non-ubiquitinated NMNAT2 (anti-FLAG) in the IPs and shown as percentage to that of the control group (Lane 2, scrambled shRNA). (C and D) Representative western blot image (C) and quantification (D) of the ubiquitination levels of NMNAT2 in N2a cells with OE of the SCFFBXO21 E3 complex (by co-expression of HA-FBXO21, HA-SKP1, HA-CUL1, and HA-RBX1). Ubiquitinated proteins are pulled down from denatured cell lysates using the His-Tag Dynabeads (for His-Ub), and the ubiquitinated NMNAT2-FLAG proteins in the His-pulldowns are detected with the anti-FLAG antibody. The relative levels of ubiquitinated NMNAT2 in the His-pulldown are normalized to the levels of NMNAT2-FLAG in the cell lysates and shown as percentage to that of the control group (Lane 3, empty HA vectors). Mean ± SEM. n = 3 in (B and D). Student’s t test; *P < 0.05 and **P < 0.01. (E) A schematic of the in vitro ubiquitination assay. The His-NMNAT2 substrate, the His-E1 and the His-E2 enzymes, as well as the StrepII-Ub are recombinant proteins expressed and purified from E. coli; the SCFFBXO21 E3 ligase complex is reconstituted from 293T cells by IP of FLAG-FBXO21 using the anti-FLAG antibody. (F) A diagram of the FBXO21 protein showing the P34A/E36A (AA) mutation within the F-box domain. (G) The expression of each subunit and the reconstitution of the SCFFBXO21-WT or SCFFBXO21-AA complex by IP from 293T cells are confirmed by western blotting. (H) The NMNAT2 protein is immunoprecipitated from the in vitro ubiquitination solution after the reaction using an anti-NMNAT2 antibody, and the ubiquitinated NMNAT2 is examined by western blotting with the UBCJ2 antibody (for mono- and poly-ubiquitinylated conjugates). #indicates the band of the heavy chain of the anti-NMNAT2 IgG. Source data are available for this figure: SourceData F5.
Figure 5.

The SCF FBXO21 E3 ligase complex promotes NMNAT2 ubiquitination in vivo and in vitro. (A and B) Representative image (A) and quantification (B) of the denaturing IP and western blot analysis of the ubiquitination levels of NMNAT2 in N2a cells with Fbxo21 KD. NMNAT2-FLAG is immunoprecipitated from denatured cell lysates with an anti-FLAG antibody, and the ubiquitinated NMNAT2 in the IPs is detected with an anti-HA antibody for (HA-Ub). Ub: ubiquitin. The relative levels of ubiquitinated NMNAT2 are normalized to non-ubiquitinated NMNAT2 (anti-FLAG) in the IPs and shown as percentage to that of the control group (Lane 2, scrambled shRNA). (C and D) Representative western blot image (C) and quantification (D) of the ubiquitination levels of NMNAT2 in N2a cells with OE of the SCFFBXO21 E3 complex (by co-expression of HA-FBXO21, HA-SKP1, HA-CUL1, and HA-RBX1). Ubiquitinated proteins are pulled down from denatured cell lysates using the His-Tag Dynabeads (for His-Ub), and the ubiquitinated NMNAT2-FLAG proteins in the His-pulldowns are detected with the anti-FLAG antibody. The relative levels of ubiquitinated NMNAT2 in the His-pulldown are normalized to the levels of NMNAT2-FLAG in the cell lysates and shown as percentage to that of the control group (Lane 3, empty HA vectors). Mean ± SEM. n = 3 in (B and D). Student’s t test; *P < 0.05 and **P < 0.01. (E) A schematic of the in vitro ubiquitination assay. The His-NMNAT2 substrate, the His-E1 and the His-E2 enzymes, as well as the StrepII-Ub are recombinant proteins expressed and purified from E. coli; the SCFFBXO21 E3 ligase complex is reconstituted from 293T cells by IP of FLAG-FBXO21 using the anti-FLAG antibody. (F) A diagram of the FBXO21 protein showing the P34A/E36A (AA) mutation within the F-box domain. (G) The expression of each subunit and the reconstitution of the SCFFBXO21-WT or SCFFBXO21-AA complex by IP from 293T cells are confirmed by western blotting. (H) The NMNAT2 protein is immunoprecipitated from the in vitro ubiquitination solution after the reaction using an anti-NMNAT2 antibody, and the ubiquitinated NMNAT2 is examined by western blotting with the UBCJ2 antibody (for mono- and poly-ubiquitinylated conjugates). #indicates the band of the heavy chain of the anti-NMNAT2 IgG. Source data are available for this figure: SourceData F5.

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