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. Author manuscript; available in PMC: 2022 Jun 16.
Published in final edited form as: J Immunol. 2017 Jul 31;199(5):1846–1855. doi: 10.4049/jimmunol.1601493

FIGURE 5. PACT interacts with MDA5, and its activation of MDA5 requires dsRNA binding.

FIGURE 5.

(A) HEK293 cells were transfected with MDA5 and PACT expression plasmids for 48 h. Total lysate was subjected to pull-down with poly(I:C)- or polyC-conjugated agarose beads. Bound fraction and input were analyzed by Western blotting. (B) A poly(I:C) pull-down experiment was carried out with endogenous MDA5 and PACT. HEK293 cells were mock treated or treated with rIFN-β (1000 U/ml) for 24 h before harvest. (C and D) HEK293 cells were transfected with MDA5 and PACT expression plasmids, alone or in combination, for 30 h before poly(I:C) stimulation (1 μg/ml). Coimmunoprecipitation was carried out with anti-V5 (C) or anti-FLAG (D) Ab. (E) HEK293 cells carrying a stably expressed IFN-β–luc were transfected with expression plasmids for MDA5 and either PACT-WT or its dsRNA-binding defective mutant (PACT-M1, PACT-M2, or PACT-DM) for 24 h, followed by poly(I:C) induction (1 μg/ml) for 6 h. Protein expression was validated by Western blotting (inset). *p < 0.01 versus PACT-WT, Student t test.