Figure 3. TRIM21 catalyzes Lys48‐linked ubiquitination of SIRT5.

• A
  In vivo ubiquitination assay analysis of WCLs and GST‐tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 h before harvesting.
• B
  IB analysis of WCLs and anti‐HA immunoprecipitates of 293T cells transfected with the indicated plasmids. 48 h post‐transfection, cells were treated with 10 μM MG132 for 4 h before harvesting.
• C
  Immunopurified SIRT5 proteins were incubated with purified recombinant E1 and ubiquitin (Ub) or mutant Ub‐K48 or Ub‐K63 in the presence or absence of purified E2 (UBE2D2) and/or TRIM21 proteins as indicated in 30°C for 90 min. The ubiquitination reaction products were resolved by SDS‐PAGE and probed with the indicated antibodies.
• D
  Immunopurified SIRT5 proteins were incubated with purified recombinant TRIM21, E1 and E2 (UBE2D2), and ubiquitin (Ub) or mutant Ub‐K48R or Ub‐K63R proteins as indicated for 45 min at 30°C. The ubiquitination reaction products were resolved by SDS‐PAGE and probed with the indicated antibodies.
• E
  WCLs from TRIM21 ^+/+ and TRIM21 ^−/− BMDMs treated with 100 ng/ml LPS for 4 h were immunoprecipitated with anti‐SIRT5 antibody and analyzed by Western blotting using antibodies specifically against Ub‐K48 and Ub‐K63. 10 μM MG132 was added for 4 h before harvesting.

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.