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. 2022 Jun 30;23(9):e54391. doi: 10.15252/embr.202154391

Figure 6. SIRT5 suppresses TRIM21 activity via deacetylation of TRIM21 at Lys351.

Figure 6

  • A
    IB analysis of WCLs and anti‐Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids using anti‐Flag, anti‐Acetyllysine, anti‐Succinyllysine, anti‐Glutaryllysine, and anti‐Malonyllysine antibodies.
  • B
    IB analysis of WCLs and anti‐TRIM21 immunoprecipitated products derived from 293T cells transfected with control or SIRT5 siRNA using anti‐Flag, anti‐acetyl Lysine (AcK), anti‐succinyl Lysine (SulK), anti‐glutaryl Lysine (GluK), and anti‐malonyl Lysine (MalK) antibodies.
  • C
    IB analysis of WCLs and anti‐Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 h before harvesting.
  • D
    IB analysis of WCLs and anti‐TRIM21 immunoprecipitated products derived from BMDMs treated 100 ng/ml LPS or left untreated for 4 h. 10 μM MG132 was added for 4 h before harvesting.
  • E
    Protein expression in SIRT5+/+ and SIRT5−/− BMDMs was determined by Western blot analysis.
  • F, G
    IB analysis of WCLs and anti‐Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 h before harvesting.
  • H
    IB analysis of WCLs and anti‐GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids in the presence or absence of 3 mM NAM for 24 h. 10 μM MG132 was added for 4 h before harvesting.
  • I
    Purified TRIM21, TRIM21‐K351R and TRIM21‐K385R proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and SIRT5 proteins as indicated at 30°C for 90 min. The ubiquitination reaction products were resolved by SDS‐PAGE and probed with the indicated antibodies.
  • J
    IB 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.