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. 2022 Nov 17;24(1):e55387. doi: 10.15252/embr.202255387

Figure 7. CUL3‐KLHL22 directly promotes the ubiquitination and degradation of IRF7.

Figure 7

  • A
    The list of ubiquitination (Ub)‐related proteins that interact with IRF7 according to the MS results.
  • B
    HEK293T cells were transfected with IRF7 and the indicated ubiquitin‐related expression plasmid. IB was performed to detect FLAG, followed by IP with an anti‐hyaluronic acid (HA) antibody.
  • C, D
    HEK293T cells were transfected with IFN‐β luciferase reporters and the indicated expression plasmids. Luciferase assays were performed to determine the fold changes with respect to the empty vector group.
  • E
    qRT–PCR analysis of IFN‐I in WT and KLHL22‐KO MEFs stimulated with polyI:C or SeV. The data are presented as the fold change relative to the Actb mRNA level.
  • F
    Immunoblot analysis of SeV‐induced IRF7 in whole‐cell lysates (WLs) of WT or KLHL22‐KO MEFs.
  • G
    After treatment with MG132, IRF7 was isolated by IP from WLs of KLHL22‐KO MEFs and subjected to IB using anti‐K48‐ubiquitin. Total cell lysates were also subjected to direct IB.
  • H
    HEK293T cells were transfected with IRF7 and FLAG‐K48R‐ubiquitin or FLAG‐ubiquitin in the presence (+) or absence (−) of KLHL22 expression plasmids. HA‐tagged IRF7 was isolated by IP, and the ubiquitination level was then measured by IB.
  • I
    HEK293T cells were transfected with IRF7 and distinct KLHL22 truncation mutants. IB of FLAG was performed, followed by IP with an anti‐HA antibody.
  • J
    HEK293T cells were transfected with KLHL22, FLAG‐K48‐ubiquitin, and various IRF7 point mutants. After treatment with MG132, IB of K48‐Ub was performed, followed by IP with an anti‐HA antibody.
  • K
    HEK293T cells were transfected with IFN‐α4 luciferase reporters and the indicated expression plasmids. Luciferase assays were performed to determine fold changes with respect to the empty vector group.

Data information: The data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of differences was determined by t‐test. *P < 0.05, **P < 0.01, and ***P < 0.005.

Please see Appendix Fig S5 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure.

Source data are available online for this figure.