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. 2021 Jan 4;282(44):32208–32221. doi: 10.1074/jbc.M704870200

FIGURE 6.

FIGURE 6

SARS-CoV PLpro interacts with IRF-3.A, HeLa PLpro-4 cells were cultured to repress or induce PLpro-TM expression and subsequently mock-infected or infected with SeV for 16 h. Left panels, expression of p396-IRF-3, total IRF-3, PLpro, and actin proteins in cell lysates were determined by immunoblot (IB) analysis. Right panels, cell lysates were subjected to immunoprecipitation (IP) with a rabbit anti-IRF-3 antibody (lanes 1-4) or a control rabbit serum (lanes 5-8). The immunoprecipitates were separated on SDS-PAGE, followed by immunoblot analysis of IRF-3 (using an mAb anti-IRF-3) and PLpro (using an mAb anti-V5 tag). B, HEK293 cells were transfected with WT (lanes 1-3), C1651A (lanes 4-6), or D1826A (lanes 7-9) PLpro-TM, respectively, and, where indicated, with an empty vector (lanes 1, 4, and 7), or a vector expressing FLAG-IRF-3 (lanes 2, 5, and 8) or untagged IRF-3 (lanes 3, 6, and 9). Cell lysates were immunoprecipitated with anti-V5 (PLpro-TM), followed by immunodetection of FLAG-IRF-3 (anti-FLAG) or PLpro-TM (anti-V5) (right panels). Asterisk denotes IgG heavy chain. Immunoblot analysis of input for FLAG-IRF-3 and PLpro-TM is shown in left panels. All three forms of PLpro-TM interacted with IRF-3. C, HEK293 cells were transfected with WT (lanes 1 and 2), C1651A (lanes 3 and 4), or D1826A (lanes 5 and 6) PLpro-Sol, respectively, and, where indicated, with an empty vector (lanes 1, 3, and 5) or a vector expressing FLAG-IRF-3 (lanes 2, 4, and 6). Cell lysates were immunoprecipitated with anti-V5 (PLpro-Sol), followed by immunodetection of FLAG-IRF-3 (anti-FLAG) or PLpro-Sol (anti-V5) (lower panels). Immunoblot analysis of input for FLAG-IRF-3 and PLpro-Sol is shown in upper panels. Note that WT and D1826A PLpro-Sol interacted with IRF-3, whereas C1651A PLpro-Sol did not. D, HeLa PLpro-4 cells induced for PLpro-TM expression were transfected with GFP, GFP-IRF-3, or GFP-IRF3-5D, respectively. Cell lysates were subjected to co-IP experiments using either GFP antibody or V5 antibody for immunoprecipitation, followed by immunoblot analysis of the immunoprecipitates using anti-GFP or anti-V5 antibodies. Note that both GFP-IRF3 and GFP-IRF3-5D were associated with PLpro-TM (detected by anti-V5), whereas free GFP was not.