American Society of Hematology

Figure 7

$Figure 7. Both USP7 and ICPO deubiquitinate TRAF6 and IKKγ. (A,B) 293T cells coexpressing FLAG-tagged TRAF6 or IKKγ, HA-ubiquitin, plus one of the following plasmids: empty vector (EV), wt ICP0, ICP0-FXE, ICP0-NLS-MUT, ICP0-M4 (A) or USP7, USP7-C223S (B). Thirty-six hours later, the cells were lysed and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. Whole-cell lysates (WCLs) were immunoblotted for ICP0 and USP7 expression. (C) HEK293-TLR2/6 cells were transfected with nonsilencing shRNA or shRNA targeting USP7 and knockdown was confirmed by WB. Forty-eight hours later, the cells were transfected with ICP0 and NF-κB-Luc reporter and stimulated with Pgn for 6 hours before luciferase activity was assayed. *P < .05. (D) 293T cells were transfected with nontargeting shRNA or shRNA-USP7. Forty-eight hours later, the cells were transfected with FLAG-TRAF6 or IKKγ, HA-ubiquitin, and ICP0 before they were lysed, and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. WCLs were immunoblotted with specific antibodies to confirm ICP0 expression and USP7 knockdown. (E) THP1 cells were transduced with HF-HSV amplicon or HSV helper virus or left untransduced. Twenty minutes later the cells were lysed, and endogenous TRAF6 and IKKγ were immunoprecipitated with anti-TRAF6/IKKγ antibodies and blotted with antiubiquitin to assess their ubiquitination status. WCLs were immunoblotted for ICP0 expression. Cell lysates were also fractionated into nuclear and cytoplasmic fractions and blotted for USP7 expression. (F) Overexpression of ICP0 does not deplete endogenous TRAF6 or IKKγ: 293T cells were transfected with increasing concentration of ICP0 together with either FLAG-tagged TRAF6 or IKKγ in the presence or absence of the proteasome inhibitor, MG132 (5 μM added for the last 12 hours), and 24 hours later, cell lysate was blotted using anti-FLAG mAb to assess ICP0 effect on TRAF6 and IKKγ. ICP0 did not deplete either TRAF6 or IKKγ. (G) Comparison of wt-USP7 and USP7.NES ability to deubiquitinate TRAF6 and IKKγ and suppress TLR-induced NF-κB response: 293T cells coexpressing FLAG-tagged TRAF6 or IKKγ, HA-ubiquitin, and USP7 or USP7-NES. Thirty-six hours later, the cells were lysed and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. (H) Enhanced deubiquitinating efficacy of USP7-NES against TRAF6/IKKγ compared with wt-USP7 correlated with its ability to suppress TLR-2/6 response to Pgn stimulation, measured as TNF-α mRNA. Error bars represent SEM.$

Both USP7 and ICPO deubiquitinate TRAF6 and IKKγ. (A,B) 293T cells coexpressing FLAG-tagged TRAF6 or IKKγ, HA-ubiquitin, plus one of the following plasmids: empty vector (EV), wt ICP0, ICP0-FXE, ICP0-NLS-^MUT, ICP0-M4 (A) or USP7, USP7-C223S (B). Thirty-six hours later, the cells were lysed and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. Whole-cell lysates (WCLs) were immunoblotted for ICP0 and USP7 expression. (C) HEK293-TLR2/6 cells were transfected with nonsilencing shRNA or shRNA targeting USP7 and knockdown was confirmed by WB. Forty-eight hours later, the cells were transfected with ICP0 and NF-κB-Luc reporter and stimulated with Pgn for 6 hours before luciferase activity was assayed. *P < .05. (D) 293T cells were transfected with nontargeting shRNA or shRNA-USP7. Forty-eight hours later, the cells were transfected with FLAG-TRAF6 or IKKγ, HA-ubiquitin, and ICP0 before they were lysed, and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. WCLs were immunoblotted with specific antibodies to confirm ICP0 expression and USP7 knockdown. (E) THP1 cells were transduced with HF-HSV amplicon or HSV helper virus or left untransduced. Twenty minutes later the cells were lysed, and endogenous TRAF6 and IKKγ were immunoprecipitated with anti-TRAF6/IKKγ antibodies and blotted with antiubiquitin to assess their ubiquitination status. WCLs were immunoblotted for ICP0 expression. Cell lysates were also fractionated into nuclear and cytoplasmic fractions and blotted for USP7 expression. (F) Overexpression of ICP0 does not deplete endogenous TRAF6 or IKKγ: 293T cells were transfected with increasing concentration of ICP0 together with either FLAG-tagged TRAF6 or IKKγ in the presence or absence of the proteasome inhibitor, MG132 (5 μM added for the last 12 hours), and 24 hours later, cell lysate was blotted using anti-FLAG mAb to assess ICP0 effect on TRAF6 and IKKγ. ICP0 did not deplete either TRAF6 or IKKγ. (G) Comparison of wt-USP7 and USP7.NES ability to deubiquitinate TRAF6 and IKKγ and suppress TLR-induced NF-κB response: 293T cells coexpressing FLAG-tagged TRAF6 or IKKγ, HA-ubiquitin, and USP7 or USP7-NES. Thirty-six hours later, the cells were lysed and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. (H) Enhanced deubiquitinating efficacy of USP7-NES against TRAF6/IKKγ compared with wt-USP7 correlated with its ability to suppress TLR-2/6 response to Pgn stimulation, measured as TNF-α mRNA. Error bars represent SEM.

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