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. 2022 Oct 10;13:5973. doi: 10.1038/s41467-022-33671-5

Fig. 4. ARIH1 promotes the oligomerization and activation of cGAS.

Fig. 4

a A scheme showing cGAMP activity assay. The treated cells were harvested and homogenized in 1 mL sterile water followed by heating at 95 °C for 10 min. The homogenates were centrifuge at 40,000 × g for 2 h and the supernatants (900 μL) were mixed with 10 × digitonin (100 μL). The diluted supernatants were incubated with single-layered HFF cells or L929 cells (2 × 105). Half an hour later, the supernatants were removed and full medium was added to culture HFFs or L929 cells for 4 h followed by qRT-PCR assays. b Cre-ER and Cre-ER;Arih1fl/fl MLFs were treated with 4-hydroxytamoxifen (4OH Tam, 1 μM) for 3 days followed by transfection with HSV120 (1 μg) for 4 h. The cells were treated as in (a) and the supernatants were used to treat HFF cells for 6 h followed by qRT-PCR analysis. c Arih1fl/fl and Lyz2-Cre;Arih1fl/fl BMDCs were transfected with HSV120 (1 μg) for 4 h. The cell were treated as in (a) and the supernatants were used to treat L929 cells for 6 h followed by qRT-PCR analysis. d 4OHT-treated Cre-ER;Arih1fl/fl MLFs treated were reconstituted with empty vector (Vec), ARIH1, or ARIH1(C357S) and transfected with HSV120 (1 μg) for 4 h. The cell cells were treated as in (a) and the supernatants were used to treat HFF cells for 6 h followed by qRT-PCR analysis. e Immunoblot assays with SDD-AGE to analyze the aggregation of cGAS and with SDS-PAGE to analyze the expression levels of the indicated proteins in 4OHT-treated Cre-ER and Cre-ER;Arih1fl/fl MLFs (left panels), in 4OHT-treated Cre-ER;Arih1fl/fl MLFs reconstituted with empty vector (Vec), ARIH1, or ARIH1C357S (middle panels), or in THP-1 cells stably transfected with shCon or shARIH1 (right panels) followed by infection with HSV-1 for 4 h. Data are of representative of three independent experiments (b–e). Graphs show mean ± S.D. (n = 4 for (b–d), biologically independent experiments). Statistical significance was determined using two-tailed Student’s t tests in (b, c), or one-way ANOVA in d. Source data are provided as a Source Data file.