Journal: Drug Design, Development and Therapy

Article Title: Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway

doi: 10.2147/DDDT.S361748

Figure Lengend Snippet: Ginsenoside Rb1 inhibits NLRP3 inflammasome activation and the NF-κB pathway in BLM-induced pulmonary inflammation in mice on day 3. ( A ) The expression of NLRP3, ASC, caspase-1, caspase-1 p10, and pro IL-1β in lung tissues, measured using Western blotting (n=6). ( C ) Immunofluorescence co-localization of NLRP3 and CD11c. ( D ) The expression of phosphorylated IκBα and NF-κB p65 in lung tissues, measured using Western blotting (n=6). ( B ) ( E ) Quantitative analysis of the Western blots shown in ( A ) and ( D ). (n=6, *P < 0.05, **P< 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Antibodies against NF-κB p65 (10745–1), IκBα (10268–1), α-smooth muscle actin (α-SMA; 80008–1), collagen type I (Col I) (66761–1), and CD11c (17342–1), as well as secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence

Journal: Drug Design, Development and Therapy

Article Title: Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway

doi: 10.2147/DDDT.S361748

Figure Lengend Snippet: G-Rb1 inhibits BLM-induced NLRP3 inflammasome activation and the NF-κB pathway in lungs on day 21. ( A ) Representative Western blots of NLRP3, ASC, caspase-1, caspase-1 p10, and pro IL-1β expression in lung tissues (n=6). ( C ) Representative Western blots of IκBα and NF-κB p65 phosphorylation in lung tissues (n=6). ( B ) ( D ) Quantitative analysis of the Western blots shown in A and C. (n=6, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Antibodies against NF-κB p65 (10745–1), IκBα (10268–1), α-smooth muscle actin (α-SMA; 80008–1), collagen type I (Col I) (66761–1), and CD11c (17342–1), as well as secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Activation Assay, Western Blot, Expressing, Phospho-proteomics

Journal: Drug Design, Development and Therapy

Article Title: Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway

doi: 10.2147/DDDT.S361748

Figure Lengend Snippet: G-Rb1 suppresses the NF-κB pathway in macrophages whereas MCC950 has no inhibitory effect on the NF-κB pathway. ( A ) ( D ) Representative Western blots of pIκBα/IκBα and pNF-κB p65/NF-κB p65. ( B ) ( E ) Quantitative analysis of the Western blots shown in ( A ) and ( D ). ( C ) Nuclear translocation of NF-κB p65 in macrophages examined using immunofluorescence. (n≥3, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Antibodies against NF-κB p65 (10745–1), IκBα (10268–1), α-smooth muscle actin (α-SMA; 80008–1), collagen type I (Col I) (66761–1), and CD11c (17342–1), as well as secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Western Blot, Translocation Assay, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots). One-way ANOVA P < 0.0001; *P < 0.05 and ***P < 0.001 versus PBS, by Tukey’s multiple comparisons test. Data represent the mean ± SEM. (B) Schematic representation of the pscFv9 plasmid used for scFv production and expression. (C) Representative Western blot of cytoplasmic and nuclear fractions of Hek293 cells. Anti-Myc antibody revealed scFv and laminin A/C or actin in the different fractions. (D) Representative image of media from transfected Hek293 cells probed with anti-Myc antibody. Ponceau staining was used as a reference. (E) Different concentrations of TDP-43 (1–206 aa, Proteintech) or BSA were loaded onto a dot blot membrane. Immunoblots were performed with media containing pscFv9-transfected Hek293 cells and E6 monoclonal antibody. Signals were revealed with anti–Myc-HRP antibody for scFv conditions or anti–mouse HRP for E6. Ponceau staining was used as a reference. (F) Representative blot of TDP-43 immunoprecipitation in pscFv9-transfected Hek293 cells. Experiments in C, D, and F were conducted more than 3 times. Empty, no scFv; CTR, control D1.3 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Binding Assay, Plasmid Preparation, Expressing, Western Blot, Transfection, Staining, Dot Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Binding of p65 to TDP-43 was measured in the presence of equal volumes of PBS or media from pscFv9-transfected Hek293cells. n = 5–8 wells per condition (dots). One-way ANOVA P = 0.002; *P < 0.05 versus PBS; #P < 0.05 or ##P < 0.001 versus control scFv by Tukey’s multiple comparisons test. (B) BV2 cells were transfected with pscFv9, stimulated with LPS or PBS, and lysated for the luciferase assay. n = 4–5 individual experiments (dots). Two-way ANOVA P = 0.0002; §§§P < 0.001 versus LPS; ***P < 0.001 versus empty; and ###P < 0.001 versus control scFv, by Tukey’s multiple comparisons test. (C and D) BV2 p65-luc cells were treated or not for 6 hours or 10 hours with scFv antibodies, together with 4 hours of LPS treatment. (C) Representative Western blots of total lysates of Myc-tag scFv inside the cells and actin (n = 3 pooled experiments). The blot of the untreated cells at 6 hours was run on the same gel but was noncontiguous. (D) NF-κB activity assessed by luciferase assay. n = 4–6 replicates (dots) from 2 independent experiments (2–3 wells each). Two-way ANOVA P < 0.0001; *P < 0.05 and ***P < 0.001 versus untreated cells; ###P < 0.001 versus control scFv, by Tukey’s multiple comparisons test. Data represent the mean ± SEM. RLU, relative luminescence units; CTR, control D1.3 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Binding Assay, Transfection, Luciferase, Western Blot, Activity Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Morinda officinalis oligosaccharides alleviate depressive‐like behaviors in post‐stroke rats via suppressing NLRP3 inflammasome to inhibit hippocampal inflammation

doi: 10.1111/cns.13732

Figure Lengend Snippet: Morinda officinalis oligosaccharides (MOOs) suppress NF‐κB signaling pathway in LPS + ATP treated primary rat microglia. (A–H) Western blot analysis of the expression of p‐IκB (B), IκB (C), p‐p65 (E), p65 (F), and nuclear p65 (np65) (H) in primary rat microglia after ATP + LPS and/or MOOs treatment ( n = 4). (I) Representative immunofluorescence images showing the translocation of p65 into nucleus. In the LPS + ATP + MOOs group, primary rat microglia were treated with MOOs at the concentration of 5 mg/mL. Scale bar, 20 μm. * p < 0.05, ** p < 0.01

Article Snippet: The following primary antibodies were used: anti‐NLRP3, 1:800, 12446, NOVUS; ASC, 1:300, 1‐78977, NOVUS; Caspase1, 1:700, A0964, AbClonal; IL‐1β, 1:700, A16288, AbClonal; IL‐18, 1:1000, 10663‐1‐AP, Proteintech; p65, 1:800, 10745‐1‐AP, Proteintech; p‐p65, 1:400, AP0475, AbClonal; IkBα, 1:1000, A1187, AbClonal; p‐IkBα, 1:500, AP0707, AbClonal; Actin, 1:1000, AC026, AbClonal; H3, 1:1000, AF0009, Beyotime.

Techniques: Western Blot, Expressing, Immunofluorescence, Translocation Assay, Concentration Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Morinda officinalis oligosaccharides alleviate depressive‐like behaviors in post‐stroke rats via suppressing NLRP3 inflammasome to inhibit hippocampal inflammation

doi: 10.1111/cns.13732

Figure Lengend Snippet: Morinda officinalis oligosaccharides (MOOs) suppress NF‐κB signaling pathway in PSD rats. (A–H) Western blot analysis of the expression of p‐IκB (B), IκB (C), p‐p65 (E), p65 (F), and nuclear p65 (np65) (H) in the hippocampus of rats treated with vehicle or MOOs ( n = 6). (I) Representative immunofluorescence images showing the translocation of p65 into nucleus in microglia. Scale bar, 15 μm. * p < 0.05, ** p < 0.01

Article Snippet: The following primary antibodies were used: anti‐NLRP3, 1:800, 12446, NOVUS; ASC, 1:300, 1‐78977, NOVUS; Caspase1, 1:700, A0964, AbClonal; IL‐1β, 1:700, A16288, AbClonal; IL‐18, 1:1000, 10663‐1‐AP, Proteintech; p65, 1:800, 10745‐1‐AP, Proteintech; p‐p65, 1:400, AP0475, AbClonal; IkBα, 1:1000, A1187, AbClonal; p‐IkBα, 1:500, AP0707, AbClonal; Actin, 1:1000, AC026, AbClonal; H3, 1:1000, AF0009, Beyotime.

Techniques: Western Blot, Expressing, Immunofluorescence, Translocation Assay