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. 2017 Mar 1;37(6):e00537-16. doi: 10.1128/MCB.00537-16

FIG 6.

FIG 6

ADAP2 mediates assembly of two modules of antiviral signaling pathway components. (A to C) ADAP2 silencing abolished interaction of MAVS with TBK1 and NEMO. (A) ADAP2 was silenced in HEK293T cells, endogenous MAVS was immunoprecipitated, and interactions with endogenous TBK1 and NEMO were probed by Western blotting. (B) ADAP2 was silenced in HEK293T cells, endogenous TBK1 was immunoprecipitated, and interactions with endogenous MAVS and NEMO were probed by Western blotting. (C) ADAP2 was silenced in human primary monocytes, endogenous MAVS was immunoprecipitated, and interactions with endogenous TBK1 and NEMO were probed by Western blotting. (D) ADAP2-mediated assembly of TBK1 with NEMO and IRF3. Overexpressed Myc-TBK1 was immunoprecipitated from ADAP2-silenced HEK293T cells, and interactions with endogenous NEMO and IRF3 were probed by Western blotting. (E) ADAP2 silencing attenuated TLR3-mediated IFN-β promoter-driven luciferase reporter activity. ADAP2-silenced TLR3-expressing HEK293T cells were stimulated by poly(I·C) added to the medium. *, P < 0.05. (F) ADAP2 silencing abolished TLR3 activation-induced interaction of endogenous TBK1 with NEMO and IRF3. Endogenous TBK1 was immunoprecipitated after poly(I·C) stimulation of TLR3, and interactions with endogenous NEMO and IRF3 were probed by Western blotting. Reporter (firefly) luciferase values were normalized with a renilla luciferase internal control and expressed as fold induction values. The values shown are means and SD for a representative experiment performed in triplicate. The statistical significance of differences was calculated by comparing the value for each test condition (gene silencing) with that for the corresponding stimulated siNT-treated control samples. si, siRNA; siNT, nontargeting negative-control siRNA; EV, empty vector; hpi, hours postinfection; SeV, Sendai virus; WCL, whole-cell lysate; IP, immunoprecipitation; IB, immunoblot; p-IRF3, phosphorylated IRF3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic internal control marker).

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