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. 2019 Apr 29;33(8):8865–8877. doi: 10.1096/fj.201802418R

Figure 5.

Figure 5

Association of ORF3a with TRAF3 and ASC. A) The TRAF binding domain is required for the association of ORF3a with TRAF2, 3, and 6 and ASC. The indicated proteins were expressed in HEK293 cells for 24 h. Cell lysates were collected for immunoprecipitation (IP). TRAF proteins were precipitated with mouse anti-FLAG. ASC was pulled down with mouse anti-ASC. Input lysates and precipitates were analyzed by Western blotting. TRAF proteins were detected with rabbit anti-FLAG. ASC was probed with rabbit anti-ASC. B) Reciprocal immunoprecipitation and Western blotting. ORF3a was expressed in HEK293 cells for 24 h. V5-tagged ORF3a was immunoprecipitated with anti-V5. TRAF3 was probed with mouse anti-TRAF3. ASC was detected with rabbit anti-ASC. C) Association of ORF3a with endogenous TRAF3 and ASC. THP-1 cells were transfected with ORF3a plasmid. Immunoprecipitation of ORF3a was performed with anti-V5 (a-V5). Precipitates were probed for endogenous TRAF3 and ASC as above. D) Colocalization of ORF3a with TRAF3 and ASC. The indicated proteins were ectopically expressed in A549 cells for 24 h. Cells were fixed and stained for the overexpressed proteins using anti-tag antibodies. TRAF3 was stained with anti-FLAG. ORF3a was probed with anti-V5. ASC was detected with anti-T7. Fluorescent signals of different colors were merged with nuclear morphology in blue revealed by DAPI staining. Transfected cells are highlighted by arrows. Scale bar, 20 µm.