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. Author manuscript; available in PMC: 2020 Jul 13.
Published in final edited form as: Nat Immunol. 2020 Jan 13;21(2):158–167. doi: 10.1038/s41590-019-0569-9

Figure 4: PolyQ protein disrupts TOLLIP:STING interaction and results in STING protein turnover in vitro and in vivo.

Figure 4:

(A) Immunoblot analysis of HEK293T cells transfected with the same amount HTTq74 plasmid and increasing amount of FLAG-TOLLIP plasmids (as indicated on top).

(B) Immunoblot analysis of HEK293T cells transfected with the same amount Flag-STING plasmid and increasing amount of HTTq74 plasmids (as indicated on top).

(C) Immunoblot analysis of STING and HTTq74 co-expression treated overnight with MG-132 (5 μM) and BafA1 (0.5 μM).

(D) IFNβ-Luciferase assay of cGAS-STING activation after dose titration of HTTq74 protein. A representative experiment with 2 biological replicates is showing. Experiments were repeated twice.

(E) Co-immunoprecipitation of STING and TOLLIP in HEK293T cells. HEK293T cells were transfected with indicated plasmids (top) in the presence of BafA1 (0.5 μM), and 24 h later anti-FLAG antibody was used to pull down FLAG-TOLLIP. HA-STING co-IP was analyzed by immunoblot. Note, BafA1 was used to prevent STING degradation caused by polyQ protein.

(F) STING protein and mRNA analysis in zQ175 knock-in mouse brain tissues. Wild type, zQ175/+ and zQ175/zQ175 mouse brain striatum and cortex were freshly dissected and processed for immunoblot and qRT-PCR analysis. n=3 mice per group. STING protein blots are showing on the left, densitometry quantitation of STING/TUBULIN band ratio is showing in the middle, and STING mRNA level (normalized to GAPDH) is showing on the right. For panel F, **, P < 0.01; n.s., not significant. Error bars represent the SEM. Unpaired Student’s t test (2-sided).

Data are representative of at least two independent experiments.