Journal: Frontiers in Pharmacology

Article Title: Amanita muscaria extract potentiates production of proinflammatory cytokines by dsRNA-activated human microglia

doi: 10.3389/fphar.2023.1102465

Figure Lengend Snippet: AME-1 upregulates TLR3 expression. (A) HMC3 cells were treated with AME-1 (50–5,000 μg/mL) for 3 h, RNA was extracted and analyzed via RT-qPCR. Results are presented as fold-gene expression relative to untreated cells and statistics were calculated using One-Way ANOVA with Dunnett’s multiple comparison post hoc analysis relative to fold-change in GAPDH expression: p ≤ 0.01 (**). Data presented as mean ± SEM for n = 3. HMC3 were treated with AME-1 (50–5,000 µg/mL), LPS (1 µg/m) or poly(I:C) (0.5 µg/mL) for 24 h, the cells were lysed and analyzed via Western blot. (B) TLR3 and IRF3 protein expression and (E, F) corresponding densitometry analysis. (C, D) MDA5 and RIG-I protein expression and (G, H) corresponding densitometry analysis. Densitometry analysis was performed on three blots on the indicated bands and statistical significance was calculated using one-way ANOVA with Tukey post hoc analysis.

Article Snippet: The membrane was subsequently blocked for 1 h at room temperature with 5% skim milk in TBS-T followed by incubation for1hr with 0.4 µg/mL of mouse anti-human TLR3 (sc-32232, Santa Cruz Biotechnology, Dallas, TX, United States), 0.5 µg/mL of mouse anti-human IRF3 (550428, BD biosciences), 2.5 µg/mL of rabbit anti-human MDA5 (33H12L34, Invitrogen), 1 µg/mL rabbit anti-human RIG-1 (PA510297, Invitrogen), or 1 µg/mL of mouse anti-β-actin loading control (PA1-183, Invitrogen) in 3% BSA-TBS-T, and finally incubated for 1 h with goat anti-mouse IRDye 680 (Li-Cor, Lincoln, NE, United States) or goat anti-rabbit IRDye 800 (Li-Cor) diluted 1:15,000 in 3% BSA-TBS-T.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

doi: 10.1371/journal.pone.0020240

Figure Lengend Snippet: Cells were stimulated with 1.0 µg/ml of P. gingivalis LPS, 0.1 µg/ml of E. coli LPS or 0.1 µg/ml of Pam3CSK4 for 12 hrs. Phosphorylation of IRF3 cellular extracts was analyzed by western blotting. Blot is a representative of three independent experiments.

Article Snippet: Rabbit anti-mouse IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho-IRF3 (Cell Signaling Technology, Danvers, MA), rabbit anti-mouse GAPDH (Santa Cruz Biotechnology), peroxidase labeled anti-rabbit antibody (GE Healthcare, Munich, Germany) and ECL Plus Western Blotting Reagent Pack (Amersham Biosciences, Buckinghamshire, UK) were used for western blotting experiments.

Techniques: Phospho-proteomics, Western Blot

Journal: Molecular cell

Article Title: A Quantitative Genetic Interaction Map of HIV Infection

doi: 10.1016/j.molcel.2020.02.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-IRF7 , Cell Signaling , 13014S, RRID:AB_2737060.

Techniques: Virus, Luciferase, Mutagenesis, Recombinant, Purification, esiRNA, Cloning, Synthesized, Software

Journal: EBioMedicine

Article Title: A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology

doi: 10.1016/j.ebiom.2021.103314

Figure Lengend Snippet: ISD017 associates with STING and STIM1 and prevents ER-to-Golgi Trafficking . (a) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml). One h later, the cells were transfected with dsDNA (2 µg/ml). Cells were lysed at the indicated time points post-stimulation and immunoblotted by antibodies against pTBKs (S172), pSTING (S366), pIRF3 (S396), and Vinculin. ( n = 3). (b) PMA-differentiated THP1 cells were pretreated with ISD017 (200 µg/ml) for 1 h. The cells were transfected with dsDNA (6 µg/ml) and fixed 2 h later for imaging analysis after staining with anti-STING, anti-Calreticulin (ER), and anti-GM130 (Golgi) ( n = 3). Scale bar, 10 µm. (c) Lysates from PMA-differentiated THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies binding the indicated proteins ( n = 3). The light blue color of the bands for STEEP and vinculin in the input lane illustrates saturation of the signal. (d) Lysates from PMA-differentiated Wt, STING KO, and STIM1 KO THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies against STIM1, STING, and vinculin ( n = 3). (e) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml) for 1 h, followed by stimulation with dsDNA for 2 hs. Cell lysates were subjected to immunoprecipitation with anti-STING and immunoblotting with anti-STIM1, anti-STING, and anti-vinculin ( n = 3). (f,g) Wt and STIM1 KO THP1 macrophages were treated with dsDNA in the presence or absence of ISD017 (200 µg/ml, pre-treatment). Supernatants were harvested 16 h post-stimulation, and IFN-I activity and TNFα levels were measured ( n = 3). The data in panel f and g are presented as means of biological triplicates +/- st.dev. [Statistical analysis of the data in f, g were performed using two-tailed one-way ANOVA test].

Article Snippet: The following antibodies and dilutions were used for immunoblotting anti‐STING (Cell Signaling, D2P2F/#13,647, 1:1000), sheep anti‐STING (R&D Systems, AF6516, 1:500), rabbit anti‐phosphoSTING (S366) (Cell Signaling Technology, #85,735, 1:1000), rabbit anti‐TBK1 (Cell Signaling, 3504, 1:1000), rabbit anti‐phospho-TBK1 (Ser172) (Cell Signaling, D52C2/#5483, 1:1000), IRF3 (Cell signaling, 11,904, 1:1000), phospho-IRF3 (S396) (Cell Signalling, 4947,1:1000, mouse anti‐p62 (Cell Signaling, # 88,588, 1:1000), anti-STEEP (Proteintech, 24,021–1-AP, 1:1000), anti-STIM1 (D88E10, Cell Signaling, #5668, 1:1000), rabbit anti‐LC3 (Cell Signaling, #3868, 1:1000), rabbit anti-cleaved caspase3 (Cell Signaling, #9664,1:1000), and mouse anti‐vinculin (Sigma, #V9131, 1:10,000).

Techniques: Transfection, Imaging, Staining, Incubation, Binding Assay, Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test