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. 2014 Feb 10;211(2):313–328. doi: 10.1084/jem.20122844

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© 2014 Fan et al.

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Figure 3. — USP21 but not A20 and CYLD binds to and deubiquitinates RIG-I-CARD. (A) FLAG-RIG-I-CARD and HA-Ub were co-transfected with MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20. The heated cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies. (B) HEK293T cells were transfected with MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20. MYC-tagged proteins were immunoprecipitated with anti-MYC antibodies. Supernatant containing polyubiquitinated FLAG-RIG-I-CARD proteins were co-incubated with the immunoprecipitated MYC-tagged proteins for 2 h at 30°C, and then immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies. (C) FLAG-RIG-I-CARD was co-transfected into HEK293T cells with control, MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20. Cell lysates were immunoprecipitated with anti-MYC antibodies and immunoblotted with anti-FLAG antibodies. Data from A–C are representative of three independent experiments.