1. Volume 35, Issue 4, Pages 647-648 (October 2011)
Mapping a Dynamic Innate Immunity Protein Interaction Network Regulating Type I Interferon Production 
Shitao Li, Lingyan Wang, Michael Berman, Young-Yun Kong, Martin E. Dorf 
Immunity 
Volume 35, Issue 4, Pages (October 2011)
DOI: /j.immuni
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 6
Mind Bomb E3 Ligases Regulate Innate Antiviral Immunity through TBK1 Ubiquitination
(A) Lysates from Mib1f/f and Mib1−/− cells (Mib1f/f MEFs infected with Adeno-Cre for removal of the floxed allele) were infected with Sendai virus, and after 2 hr, cell lysates were immunoprecipitated with control IgG or TBK1 antibody and immunoblotted with MIB1 or NAP1 antibody to examine endogenous interactions induced by Sendai virus infection.
(B) Sendai virus induces TBK1 ubiquitination. HEK293 cells were treated with 50 HA Sendai virus for the designated times. The immune complexes were immunoblotted with TBK1 or antibody specific for K63-linked ubiquitin.
(C) In vitro ubiquitination reaction of TBK1 in the presence of MIB1, MIB2, or MIB2 mutant.
(D) Mib1f/f and Mib1−/− cells were transfected with MIB2 siRNA (Mib2KD). 48 hr later, cells were treated with 50 HA Sendai virus for the designated times. Cell lysates were immunoprecipitated and immunoblotted with TBK1 antibody.
(E) Mouse embryonic fibroblasts (MEFs) from Mib1f/f and Mib1−/− mice were transfected with control or MIB2 siRNA. After 48 hr, cells were treated with indicated stimulus for the designated times. Cell lysates or nuclear extracts were collected for immunoblotting with the indicated antibodies.
(F) MEFs from Mib1f/f and Mib1−/− mice were transfected with control or MIB2 siRNA. After 48 hr, cells were treated with indicated stimulus. Cytokine mRNA levels were determined by real-time PCR. Data represent the mean ± SD of triplicate samples. ∗p < 0.05.
(G) Bone marrow-derived macrophages (BMDM) from Mib2+/− and Mib2−/− mice were transfected with IKKε siRNA together with the indicated siRNA. After 48 hr, cells were infected with Sendai virus for the designated times. Cytokine mRNA expression was determined by real-time PCR. ∗p < Data depict the mean ± SD of triplicate samples.
(H and I) BMDM from Mib2+/− and Mib2−/− mice were transfected with IKKε siRNA together with the indicated siRNA. After 48 hr, cells were treated with Sendai virus, HSV, and LPS. IFN-β and IL-6 protein levels were determined by ELISA. Data represent the mean ± SD of triplicate samples. ∗p < 0.05.
(J) VSV-Luc replication in HEK293 cells expressing control (cross-hatched), MIB1 (open), MIB2 (checkered), or MIB2 mutant (solid) constructs. Cells were infected with the indicated amount of virus for 24 hr. Data represent the mean ± SD of triplicate samples.
(K) VSV-Luc replication in Mib1f/f (solid), Mib1−/− (cross-hatched), and Mib1−/− MEFs transfected with MIB2 siRNA (open) or control (checkered). Cells were infected with the indicated dose of VSV. Data represent the mean ± SD of triplicate samples.
(L) VSV-eGFP replication in Mib1f/f, Mib1−/−, or Mib1−/− MEFs treated with control or MIB2 siRNA. Upper panel, green fluorescence; lower panel, bright field.
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions