Figure EV3. HAUSP physically interacts with SIRT5.

• A
  IB analysis of WCLs and immunoprecipitates derived from 293T cells transfected with Flag‐HAUSP together with HA‐SIRT5 constructs. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• B
  Purified GST‐SIRT5 proteins were incubated with purified recombinant Flag‐HAUSP proteins as indicated for 90 min at 30°C, followed by pull down with anti‐GST antibody and Western blot analysis.
• C
  IB analysis of WCLs and IPs derived from 293T cells transfected with Flag‐HAUSP together with the indicated SIRT5 deletion constructs. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• D
  IB analysis of WCLs and immunoprecipitates derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• E
  IB analysis of WCLs and immunoprecipitates derived from 293T cells transfected with Flag‐HAUSP together with HA‐SIRT5 constructs in the presence or absence of 3 mM NAD⁺ or NADH as indicated. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• F
  IB analysis of WCLs and immunoprecipitates derived from 293T cells transfected with Flag‐HAUSP together with HA‐SIRT5, HA‐SIRT5‐K0, HA‐SIRT5‐10KR as indicated. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• G
  293T cells were transfected with increasing amounts of Flag‐HAUSP construct and endogenous SIRT5 expression was analyzed by Western blotting.
• H
  IB analysis of WCLs and immunoprecipitates derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 4 h before harvesting.
• I
  mRNA levels of HAUSP and SIRT5 in BMDMs transfected with control siRNA (−) or HAUSP siRNA for 48 h were measured 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.

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