Journal: Frontiers in Neuroscience

Article Title: Modeling Alzheimer’s disease related phenotypes in the Ts65Dn mouse: impact of age on Aβ, Tau, pTau, NfL, and behavior

doi: 10.3389/fnins.2023.1202208

Figure Lengend Snippet: Total Tau and pTau S396 protein level in Ts65Dn and 2N mouse brain total homogenate and insoluble fraction at different ages by Meso Scale Discovery and WB. (A) Schematic representation of the fractions used for evaluation. The total homogenate was used for measuring total Tau and pTau levels. P3-SinT was used for the Sarkosyl insoluble Tau fraction. (B) Total Tau measured by Meso Scale Discovery. Age had a significant effect on levels of total Tau ( p < 0.0001). (C) Total Tau as measured by WB. Age had a significant effect ( p = 0.0218) as did genotype ( p = 0.0218). (D) pTau S396 protein level in total brain homogenates of 2N and TS65Dn mice at different ages as measured by Meso Scale Discovery. Age ( p < 0.0001) and genotype ( p = 0.0228) were significant factors for pTau S396 levels. (E) WB evaluation of pTau S396 protein level in total brain homogenates of 2N and TS65Dn mice at different ages measured. Age ( p < 0.0001) was a significant factor for pTau S396 levels. The concentrations of pTauS396 obtained by Meso Scale Discovery were normalized on the total protein concentration, while the results obtained by WB were normalized on the total protein concentration and expressed as the corrected area. (F) pTau S396 protein level in Sarkosyl-insoluble fraction of the 13- and 16-month-old cohort measured by WB. ANOVA indicated a significant effect of genotype ( p = 0.0066), age ( p = 0.0024), and interaction of age and genotype ( p = 0.0066). (G) pTau S396 protein level in Sarkosyl-insoluble fraction of the 16-month-old cohort measured by Meso Scale Discovery. Results are expressed as individual values and mean ± SD.

Article Snippet: Post hoc Tukey’s multiple comparisons tests identified significant increases in pTau S396 levels measured by Meso Scale Discovery in Ts65Dn mice at 10 ( p < 0.0001), 13 ( p < 0.0001), and 16 ( p < 0.0001) months of age compared to 7 months of age, and at 16 months of age compared to 10 months of age ( p = 0.0053).

Techniques: Protein Concentration

Journal: Frontiers in Immunology

Article Title: Foot-and-mouth disease virus non-structural protein 2B downregulates the RLR signaling pathway via degradation of RIG-I and MDA5

doi: 10.3389/fimmu.2022.1020262

Figure Lengend Snippet: FMDV 2B inhibit the transcription of antiviral genes and type-I IFN signaling. Control vector and FMDV 2B stably expressing RAW264.7 cells were infected with PR8-GFP. Cells were harvested at indicated time points after the infection of PR8-GFP. Total and phosphorylated TBK1, IRF3, and p65 were measured by immunoblotting. β-actin was used as the loading control (A) . Control vector and FMDV 2B stably expressing RAW264.7 cells were infected with VSV-GFP. At 0 and 24 hpi, cells were harvested, and quantitative real-time PCR was performed to analyze the levels of antiviral genes (B) . HEK293T cells were transfected with interferon-β promoter encoding firefly luciferase plasmid, TK-Renilla plasmid, increasing dose of Flag-FMDV 2B plasmid and stimulated with H1N1, NDV infection, Poly(I:C) treatment (C) or transfected with MDA5, RIG-I, MAVS, TRIF, and TBK1 encoding plasmids (D) for 24 hours. Results are expressed relative to those of Renilla luciferase alone (internal control). Results representative of at least two independent experiments, each with similar results, and the values are expressed as mean ± SD of three biological replicates. Student’s t-test; **p < 0.01; ***p < 0.001.

Article Snippet: Antibodies used for the immunoblot and immunoprecipitation analysis are as follows, anti-Flag (Cell Signaling, 8146), anti-Strep (Qiagen, 34850), anti-GST (Santa Cruz, sc-138), anti-IRF3 (Abcam, ab25950), anti-phospho IRF3 (Ser396) (Cell Signaling, 4947), anti-p65 (Cell Signaling, 4764S), anti-phospho p65 (Cell Signaling, 3031S), anti-TBK1 (Cell Signaling, 3504S), anti-phospho-TBK1 (Cell Signaling, 5483S), anti-β-actin (Santa Cruz,SC 47778), RIG-I (D14G6; 3743), MDA-5 (D74E4; 5321), Anti-FMDV 2B (homemade), anti-Caspase8 (Cell Signaling, 9746S), anti-Caspase3 (Cell Signaling, 9662S) and anti-β-actin (Santa Cruz,SC 47778)

Techniques: Control, Plasmid Preparation, Stable Transfection, Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Luciferase