Journal: Investigative ophthalmology & visual science
Article Title: Inhibition of LRRK2 Ameliorates Aspergillus fumigatus Keratitis by Regulating STING Signaling Pathways.
doi: 10.1167/iovs.66.2.13
Figure Lengend Snippet: FIGURE 4. LRRK2 regulates STING downstream pathways. HCECs were transfected with pcDNA3.1 (vector) or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae (1 × 106 hyphal fragments/mL) for 6 hours. (A) Western blot was performed to detect the protein levels of p-TBK1, TBK1, p-IκBα, IκBα, p-p65, p65, p-IRF3, IRF3, p-LRRK2, LRRK2, and β-actin. (B) Western blot was performed to detect the protein levels of p65 in the cytoplasm and nucleus fractions. Lamin B1 and GAPDH were used to indicate cytoplasm and nucleus, respectively. (C) Immunofluorescence staining was performed to detect p65 nuclear translocation. (D) HCECs were transfected with NC siRNA or LRRK2 siRNAs (siLRRK2-1 and siLRRK2-2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (E) HCECs were transfected with NC siRNA, LRRK2 siRNA-2 (siLRRK2-2), or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (F) HCECs were pretreated with LRRK2-IN-1 (0, 1, or 2 μM) for 12 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (G) HCECs were transfected with NC siRNA, LRRK2 siRNA-2 (siLRRK2-2), STING siRNA (siSTING), or pcDNA3.1-STING (STING) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. Quantification of protein levels shown in panels A to G is provided in Supplementary Figures S4A to S4G.
Article Snippet: Antibodies against p-STING (Ser366, 19781; Ser365, 72971; 1:1000), p-TBK1 (5483, 1:1000), p-IκBα (2859, 1:1000), p-IRF3 (4947, 1:1000), p-p65 (3033, 1:1000), and p65 (8242, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).
Techniques: Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Translocation Assay
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