Figure EV2. Identification of the lysine residues in SIRT5 that are responsible for TRIM21 ubiquitination.

• A
  IB analysis of WCLs from 293T cells transfected with HA‐SIRT5 together with increasing amounts of Flag‐TRIM21 constructs.
• B
  mRNA levels of TRIM21 and SIRT5 in TRIM21 ^+/+ and TRIM21 ^−/− BMDMs were analyzed by qRT‐PCR. Data are means ± SD. n = 3 biological replicates. P‐values were determined by unpaired two‐tailed Student's t‐tests. ns, not significant.
• C
  IB analysis of WCLs and anti‐GST immunoprecipitated products derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• D
  Purified TRIM21 and its mutant proteins as indicated were incubated with purified E1, E2 (UBE2D2) and ubiquitin at 30°C for 90 min. TRIM21 ubiquitination and protein levels were analyzed by Western blotting.
• E
  Mutants of human SIRT5 in which various lysine residues (K) are substituted with arginine (R).
• F, G
  In vivo ubiquitination assay analysis of WCLs and anti‐GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 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.