TBK1 Antibody - #DF7026

Figure 6 SHP2 negatively regulates type I interferon signaling via STING–TBK1–IRF3 pathway. PMA-differentiated THP1 cells were simulated by 2′,3′-cGAMP (0.5 μg/mL) for 2, 4, and 6 h or simulated by 2′,3′-cGAMP for 4 h after incubation with or without the indicated concentrations of SHP099 for 1 h. (A, B) Relative mRNA expression of CXCL10, IFIT1, IFIT2 and ISG15 was examined by qPCR. (C, D) IFN-β in the supernatant were detected by ELISA. Primarily cultured BMDMs from WT and Ptpn11lyz2−/– were simulated by CMA (500 μg/mL) for 2, 4, or 6 h as indicated. (E) Relative mRNA expression of Cxcl10, Ifit1, Ifit2 and Isg15 were examined by qPCR. (F) IFN-β in the supernatant were detected by ELISA. Data represent mean ± SEM; (A) and (C): n = 5; (B, D–F): n = 3; ∗P < 0.05. (G) Protein levels of p-TBK1, TBK1, p-IRF3, IRF3, STING and SHP2 were analyzed by Western blot. β-Actin was shown as loading control. (H) Primarily cultured BMDMs from WT and Ptpn11lyz2−/– were simulated by CMA (500 μg/mL) for 30 and 60 min as indicated. The subcellular localization of IRF3 (shown in green) was analyzed via confocal microscopy. Cell nuclei were visualized by DAPI (blue). Scale bar, 10 μm. (I) Percentage of the cells with IRF3 nuclear localization from Panel H. Data are shown as the mean ± SEM of five fields of view, ∗P < 0.05.

Fig. 3 TBK1-AMPK signaling pathway modulated the omentin expression in LPS-induced 3T3-L1 adipocytes. Differentiated 3T3-L1 cells were treated with or without AICAR (500 μM) for 2 h, followed by stimulation with LPS (1 μg/mL) for 24 h. (A) Expression of the ratio of p-TBK1 to TBK1 was measured by Western blot in differentiated 3T3-L1 cells (n = 5). (B) Expression of the ratio of p-AMPK to AMPK was measured by Western blot in differentiated 3T3-L1 cells (n = 5). Quantitative real-time PCR analysis of (C) TNF-α and (D) omentin mRNA expression. (E) The omentin fluorescence intensity was detected by immunofluorescence (Scale bar = 20 μm) and (F) the expression of omentin was detected by Western blot in differentiated 3T3-L1 cells (n = 5). Differentiated 3T3-L1 cells were treated with or without compound C (CC; 10 μM) for 2 h, followed by stimulation with LPS (1 μg/mL) for 24 h. Quantitative real-time PCR analysis of (G) TNF-α, (H) omentin mRNA expression. (I) The omentin fluorescence intensity was detected by immunofluorescence (Scale bar = 20 μm) and (J) the expression of omentin protein was detected by Western blot in differentiated 3T3-L1 cells (n = 5). Differentiated 3T3-L1 cells were treated with or without amlexanox (Amx; 500 μM) for 2 h, followed by stimulation with LPS (1 μg/mL) for 24 h. Quantitative real-time PCR analysis of (K) TNF-α and (L) omentin mRNA expression in differentiated 3T3-L1 cells (n = 5). (M) The omentin fluorescence intensity was detected by immunofluorescence (n = 5). Scale bar = 20 μm. (N) The expression of omentin and (O) the ratio of p-AMPK to AMPK were detected by Western blot in differentiated 3T3-L1 cells (n = 5). (P) The effects of TBK1-siRNA on omentin mRNA expression were detected by quantitative real-time PCR analysis. Values are expressed as the means ± SD. One-way ANOVA was followed by the Dunnett's post hoc test. #P < 0.05, ##P < 0.01, ###P < 0.001 LPS vs. Control; *P < 0.05, **P < 0.01, ***P < 0.001 Rd + LPS or AICAR + LPS or CC + LPS or Amx + LPS vs. LPS.