Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Zanthoxylum bungeanum Seed Oil Attenuates LPS-Induced BEAS-2B Cell Activation and Inflammation by Inhibiting the TLR4/MyD88/NF- κ B Signaling Pathway.

doi: 10.1155/2021/2073296

Figure Lengend Snippet: Figure 5: Docking results for the active components in ZBSO with key protein molecules in the TLR4/MyD88/NF-κB signaling pathway. (a) ,e structures of active components in ZBSO were obtained by screening. (b) α-Linolenic acid, quercetin, and TLR4, MyD88, or NF- κB p65 proteins in molecular docking mode.

Article Snippet: LPS was obtained from Solarbio (055: B5, Beijing, China).,e antibodies against MyD88 (23230-1-AP), NF-κB p65 (10745-1-AP), and phospho-NF-κB p65 were obtained from Proteintech (Hubei, China).,e antibodies against TRL4 (GB11519) and β-actin were gained from Servicebio (Hubei, China).

Techniques:

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Zanthoxylum bungeanum Seed Oil Attenuates LPS-Induced BEAS-2B Cell Activation and Inflammation by Inhibiting the TLR4/MyD88/NF- κ B Signaling Pathway.

doi: 10.1155/2021/2073296

Figure Lengend Snippet: Figure 6: ZBSO inhibition of LPS-induced p65 nuclear translocation. (a) Typical immunofluorescence images of NF-κB p65 (red) and Hoechst 33258 (blue) induced by LPS (20 × magnification). Western blot analysis of NF-κB p65 total protein, NF-κB p65 phosphorylation (b), and NF-κB p65 in the nucleus (c) and cytoplasm (d). ,e values are expressed as the mean ± SD (n 3) of three independent ex- periments. #p < 0.05 and ##p < 0.01 versus the control group; ∗p < 0.05 and ∗∗p < 0.01 versus the LPS group.

Article Snippet: LPS was obtained from Solarbio (055: B5, Beijing, China).,e antibodies against MyD88 (23230-1-AP), NF-κB p65 (10745-1-AP), and phospho-NF-κB p65 were obtained from Proteintech (Hubei, China).,e antibodies against TRL4 (GB11519) and β-actin were gained from Servicebio (Hubei, China).

Techniques: Inhibition, Translocation Assay, Western Blot, Phospho-proteomics, Control

Journal: Cells

Article Title: Gas6/TAM Signalling Negatively Regulates Inflammatory Induction of GM-CSF in Mouse Brain Microglia

doi: 10.3390/cells10123281

Figure Lengend Snippet: Gas6 inhibits LPS-induced nuclear translocation of the NF-κB p65 subunit in microglia. Pure microglial cell cultures were treated with LPS (10 ng/mL) for 30 min with or without 1 h pre-incubation with Gas6 (1.6 µg/mL), which then remained throughout. ( A ) Cells underwent immunofluorescence staining with anti-p65 primary antibody with AlexaFluor647 anti-mouse secondary antibody and DAPI nuclear counterstaining. Scale bar = 100 µm. ( B ) p65 staining within the nuclear area of each cell was quantified for each treatment group. Data is shown in a violin plot with median and interquartile ranges visible ( n > 30 cells). Data was statistically analysed using one-way ANOVA; **** p < 0.0001 vs. both other conditions.

Article Snippet: For staining, coverslips were incubated in primary mouse anti-p65 antibody (1:300; Proteintech, Manchester, UK) at 4 °C overnight.

Techniques: Translocation Assay, Incubation, Immunofluorescence, Staining

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