Figure 5. HAUSP deubiquitinates SIRT5, and LPS treatment suppresses this effect in macrophages.

• 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‐GST immunoprecipitates of 293T cells transfected with the indicated plasmids in the presence or absence of 100 μM P22077 for 24 h. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• C, D
  IB 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.
• E
  In vitro SIRT5 deubiquitination assay. Ubiquitinated SIRT5 was pulled down from 293T cells cotransfected HA‐Ub, and then incubated with purified wild‐type HAUSP or the catalytically inactive HAUSP mutant (HAUSP‐CS) 30°C for 90 min. The reaction products were resolved by SDS‐PAGE and probed with the indicated antibodies.
• F
  IB analysis of WCLs and anti‐SIRT5 immunoprecipitated products derived from BMDMs transfected with control or HAUSP siRNA for 48 h. 10 μM MG132 was added for 6 h before harvesting.
• G
  BMDMs transfected with control (−) or HAUSP siRNA for 48 h were then treated with 100 ng/ml mM LPS for 4 h, and 10 μM MG132 was added for 4 h before harvesting. WCLs and anti‐SIRT5 immunoprecipitated products were prepared and subjected to IB analysis using indicated antibodies.

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