anti psting ser366  (Cell Signaling Technology Inc)

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 directly interacts with DDX41 and regulates its phosphorylation. ( A ) HCECs transfected with either pcDNA3.1 (Flag) or pcDNA3.1 PIM1 (Flag-PIM1) were stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Cell lysates were immunoprecipitated using anti-Flag beads, and bound proteins were separated by SDS-PAGE and visualized by silver staining. ( B ) A GST pull-down assay was performed to examine direct binding between PIM1 and DDX41. ( C ) Endogenous interaction between PIM1 and DDX41 in HCECs was assessed by co-IP. HEK293T cells were cotransfected with Flag-PIM1 and Myc-DDX41 for 48 hours. (D) Co-IP was carried out with anti-Flag beads, and Myc-DDX41 was detected with anti-Myc antibody. ( E ) Reciprocal co-IP was performed using anti-Myc beads, and Flag-PIM1 was probed with anti-Flag antibody. ( F ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours. DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and DDX41 antibodies. ( G ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours, followed by DDX41 immunoprecipitation and immunoblotting with indicated antibodies. ( H ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Co-IP of DDX41 was performed, and associated proteins were analyzed by Western blot. ( I ) Quantification of protein levels in ( H ). ( J ) HCECs were pretreated with SGI-1776 (0 and 4 µM) for 12 hours prior to 12-hour stimulation with A. fumigatus hyphae. Co-IP assay was performed using anti-DDX41 antibody and immunoblotted with the antibodies indicated. ( K ) Quantification of protein levels in ( J ). ( L ) Recombinant His-PIM1 and GST-DDX41 were incubated in reaction buffer with or without phosphatase for 30 minutes. The protein levels of p-Ser, His-PIM1, and GST-DDX41 were detected by Western blot. Quantification of protein levels in F, G, and L is shown in A–C. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Western Blot, Recombinant, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: Knockdown of PIM1 ameliorates A. fumigatus keratitis in mice. ( A ) Mouse corneas were infected with 5 µL of live A. fumigatus hyphae (1 × 10 8 hyphal fragments/mL) for 0.5, 1, and 3 days, then excised for analysis. Protein levels of p-STING, STING, PIM1, and β-actin were assessed by Western blot. Quantitative data are provided in A. ( B ) DDX41 was immunoprecipitated from corneal lysates and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is shown in B. Mice received subconjunctival injection of 5 µL control siRNA (siNC, 10 µM) or PIM1 siRNAs (siPIM1-1/siPIM1-2, 10 µM). After 24 hours, corneas were infected with 5 µL of live hyphae for an additional 24 hours before harvesting. ( C ) Keratitis severity was evaluated by slit-lamp examination. ( D ) Clinical scores were calculated based on slit-lamp observations. ( E ) Fungal burden was quantified by colony-forming unit (CFU) assays. ( F ) Protein expression of p-STING, STING, PIM1, and β-actin in mouse corneas was detected by Western blot. Quantitative results are shown in C. ( G ) DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is presented in D. ( H ) mRNA expression of TNF-α , IL-6 , IL-1β , and IFN-β was measured by qRT-PCR. ( I ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in corneal homogenates were detected using ELISA. Data are presented as the mean ± SD; ** P < 0.01; n = 6.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Infection, Western Blot, Immunoprecipitation, Injection, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 inhibitor inhibits A. fumigatus keratitis in vivo. Mouse corneas were infected with 5 µL of live A. fumigatus hyphae (1 × 10 8 hyphal fragments/mL) for 24 hours. Two hours prior to infection, mice received a subconjunctival injection of 5 µL SGI-1776 (1 µg/µL or 2 µg/µL). Corneas were harvested 24 hours postinfection. ( A ) Keratitis severity was evaluated by slit-lamp examination. ( B ) Clinical scores were calculated based on slit-lamp observations. ( C ) Fungal burden was quantified by CFU assays. (D) Protein expression of p-STING, STING, PIM1, and β-actin in mouse corneas was analyzed by Western blot. Quantification is presented in A. ( E ) DDX41 was immunoprecipitated from corneal lysates and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is provided in B. ( F ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were measured by qRT-PCR. ( G ) Protein levels of TNF-α, IL-6, IL-1β, and IFN-β in corneal homogenates were determined using ELISA. Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01; n = 6.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vivo, Infection, Injection, Expressing, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: Working model of the PIM1-DDX41-STING axis in A. fumigatus keratitis. A. fumigatus infection upregulates PIM1 expression. PIM1 then directly binds to and phosphorylates the innate immune sensor DDX41. Phosphorylated DDX41 activates the STING signaling pathway, leading to the phosphorylation of TBK1 and IRF3. This cascade ultimately promotes the transcription and secretion of proinflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-β), contributing to the immunopathology of fungal keratitis. Pharmacologic inhibition of PIM1 by SGI-1776 disrupts this axis, attenuating DDX41/STING activation and the subsequent inflammatory response.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Expressing, Phospho-proteomics, Inhibition, Activation Assay