b cells nf kb p65  (Cell Signaling Technology Inc)

Journal: Genes & Diseases

Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

doi: 10.1016/j.gendis.2025.101987

Figure Lengend Snippet: HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Afterward, before incubation with primary antibodies overnight at 4 °C, membranes were blocked with 5% non-fat milk at 37 °C for 1 h. Subsequently, membranes were treated with horseradish peroxidase-conjugated secondary antibodies against rabbit or mouse IgG (Abcam, Massachusetts, USA, 1:5000) at 37 °C for 1 h. The following primary antibodies were used to assess the expression of proteins: anti-β-actin (#3700; Cell Signaling Technology, Massachusetts, USA, 1:5000), anti-TGIF2 (#ab190152; Abcam, 1:1000), anti-p-TGIF2 and TGIF2 (#sc-390870; Santa Cruz, CA, USA), anti-HMGB3 (#ab75782; Abcam, 1:1000), anti-TLR3 (#ab62566; Abcam, 1:1000), anti-TGF-β (#ab215715; Abcam , 1:1000), anti-SMAD2/3 (#8685; Cell Signaling Technology, 1:1000), anti-SMAD2 (#5339; Cell Signaling Technology, 1:1000), anti-p-SMAD2 (#3108; Cell Signaling Technology, 1:1000), anti-SMAD3 (#9523; Cell Signaling Technology, 1:1000), anti-p-SMAD3 (#9520; Cell Signaling Technology, 1:1000), anti-extracellular signal-regulated kinase 1/2 (ERK1/2) (#4695; Cell Signaling Technology, 1:1000), anti-p-ERK1/2 (#4370; Cell Signaling Technology, 1:1000), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) P65 (#8242S; Cell Signaling Technology, 1:1000), and anti-His-tag (#66005-1-Ig; protein-tech, 1:1000).

Techniques: Immunohistochemistry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Immunoprecipitation, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

Journal: Pharmaceutics

Article Title: CuMV VLPs Containing the RBM from SARS-CoV-2 Spike Protein Drive Dendritic Cell Activation and Th1 Polarization

doi: 10.3390/pharmaceutics15030825

Figure Lengend Snippet: VLPs induce NF- k B nuclear translocation in human DCs. DCs were treated with 20 μg/mL Poly I:C and 2.5 μg/mL R848, or 10 μg/mL VLPs, or VLPs plus 20 μg/mL Poly I:C and 2.5 μg/mL for 24 h. ( A ) I k Bα levels were evaluated using Western blot. Each column expresses the normalized expression to the control (not stimulated cells) and represents the mean ± SEM of five independent experiments. Representative images are shown. **** p < 0.0001 relative to the CTR. ( B ) Fluorescence staining of the nuclei (blue) and immunofluorescence staining of NF-κB/p65 (green) were performed as described in Materials and Methods. Control cells (CTR) were not stimulated. Scale bar 10 µm. Representative images of each condition are shown. LPS: lipopolysaccharide; VLP: virus-like particles, PolyI:C: polyinosinic:polycytidylic acid, R848: resiquimod.

Article Snippet: Then, wells were washed once with PBS, and DCs were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three times with PBS 1% + glycine 0.1 M. Cells were then blocked for 1 h with 5% goat serum + 0.3% Triton X-100 in PBS and further incubated with the primary rabbit monoclonal anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 XP ® antibody, at the dilution of 1:400 (Cell Signaling Technology, Inc., Danvers, MA, USA), overnight at 4 °C.

Techniques: Translocation Assay, Western Blot, Expressing, Control, Fluorescence, Staining, Immunofluorescence, Virus

Journal: Scientific Reports

Article Title: Pericytes take up and degrade α-synuclein but succumb to apoptosis under cellular stress

doi: 10.1038/s41598-022-20261-0

Figure Lengend Snippet: Inflammatory responses of microglia treated with two α-syn preparations. Inflammatory responses of primary human brain microglia when treated with vehicle, ribbons, fibrils (100 nM), LPS (10 ng/mL) and IL-1β (10 ng/mL). ( A ) Representative immunocytochemistry images of ICAM-1 induction after 48 h treatment Scale bar = 200 µm. Immunocytochemistry quantification of ( B ) NF-κB nuclear translocation after 1 h treatment, ( C ) MCP-1 and ( D ) ICAM-1 induction after times indicated ( n = 3, mean ± SD ). Microglia were cultured and treated with vehicle, two different α-synuclein preparations (100 nM), LPS (10 ng/mL) and IL-1β (10 ng/mL) before conditioned media were taken and cytokine secretion analysed by cytometric bead array. Heatmap analysis of cytokine secretion of microglia after treatment at ( E ) 8 h, (F) 24 h and ( G ) 48 h ( n = 3). Microglia were cultured and treated with vehicle, two different α-synuclein preparations (100 nM), LPS (10 ng/mL) and IL-1β (10 ng/mL) for 24 h before conditioned media were taken and cytokine secretion measured using a Proteome Profiler Human XL Cytokine Array Kit. Clustered heatmap analysis ( H ) of the top 30 secretions in microglia ( n = 1, epilepsy case). #LPS, + IL-1β vs vehicle p < 0.05; one way ANOVA with Tukey’s multiple comparisons test or two way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Cells were incubated with primary antibodies—nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 (Abcam, ab32536, 1:500), PDGFRβ (Y92) (Abcam, ab32570, 1:500), intracellular adhesion molecule-1 (ICAM-1) (Santa Cruz, sc-107, 1:500), monocyte chemoattractant protein-1 (MCP-1) (Abcam, ab9669, 1:500), α-syn (Abcam, ab1903, 1:3000), cleaved caspase-3 (Abcam, ab2302, 1:500) and LAMP-1 (DHSB H4A3, 1:500) overnight at 4 °C, diluted in PBS containing 1% normal goat serum.

Techniques: Immunocytochemistry, Translocation Assay, Cell Culture

Journal: Scientific Reports

Article Title: Pericytes take up and degrade α-synuclein but succumb to apoptosis under cellular stress

doi: 10.1038/s41598-022-20261-0

Figure Lengend Snippet: Inflammatory responses of pericytes treated with two α-syn preparations. Inflammatory responses of primary human brain pericytes when treated with vehicle, ribbons, fibrils, (100 nM), LPS (10 ng/mL) and IL-1β (10 ng/mL). ( a ) Representative immunocytochemistry images of ICAM-1 induction after 48 h treatment. Scale bar = 200 µm. Immunocytochemistry quantification of ( b ) NF-κB nuclear translocation after 1 h treatment, ( c ) MCP-1 and ( d ) ICAM-1 induction after times indicated ( n = 3 epilepsy cases , mean ± SD ). Pericytes were cultured and treated with vehicle, two α-syn preparations (100 nM), LPS (10 ng/mL) and IL-1β (10 ng/mL) before conditioned media were taken and cytokine secretion analysed by cytometric bead array. Heatmap analysis of cytokine secretion of pericytes after treatment at ( e ) 8 h, ( f ) 24 h and ( g ) 48 h ( n = 3 epilepsy cases). Pericytes were cultured and treated with vehicle, two different α-synuclein preparations (100 nM), LPS (10 ng/mL) and IL-1β (10 ng/mL) for 24 h before conditioned media were taken and cytokine secretion measured using a Proteome Profiler Human XL Cytokine Array Kit. Clustered heatmap analysis ( h ) of the top 30 secretions in pericytes ( n = 1, epilepsy case). # LPS, + IL-1β vs vehicle p < 0.05; one way ANOVA with Tukey’s multiple comparisons test or two way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Cells were incubated with primary antibodies—nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 (Abcam, ab32536, 1:500), PDGFRβ (Y92) (Abcam, ab32570, 1:500), intracellular adhesion molecule-1 (ICAM-1) (Santa Cruz, sc-107, 1:500), monocyte chemoattractant protein-1 (MCP-1) (Abcam, ab9669, 1:500), α-syn (Abcam, ab1903, 1:3000), cleaved caspase-3 (Abcam, ab2302, 1:500) and LAMP-1 (DHSB H4A3, 1:500) overnight at 4 °C, diluted in PBS containing 1% normal goat serum.

Techniques: Immunocytochemistry, Translocation Assay, Cell Culture

Journal: Scientific Reports

Article Title: Pericytes take up and degrade α-synuclein but succumb to apoptosis under cellular stress

doi: 10.1038/s41598-022-20261-0

Figure Lengend Snippet: TLR ligands and α-syn treatment on pericytes. Inflammatory responses of primary human brain pericytes when treated with vehicle, ribbons (100 nM), fibrils (100 nM) and TLR ligands (Supplementary Table ). Immunocytochemistry quantification of ( a ) NF-κB nuclear translocation after 1 h treatment. Conditioned media were taken and cytokine secretion analysed by cytometric bead array after 24 h. Secretions of ( b ) ICAM-1, ( c ) VCAM-1, ( d ) Fractalkine, ( e ) IL-6, ( f ) IL-8, ( g ) MCP-1, ( h ) IP-10 and ( i ) RANTES at 24 h after treatment ( n = 5 epilepsy cases, mean ± SD).

Article Snippet: Cells were incubated with primary antibodies—nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 (Abcam, ab32536, 1:500), PDGFRβ (Y92) (Abcam, ab32570, 1:500), intracellular adhesion molecule-1 (ICAM-1) (Santa Cruz, sc-107, 1:500), monocyte chemoattractant protein-1 (MCP-1) (Abcam, ab9669, 1:500), α-syn (Abcam, ab1903, 1:3000), cleaved caspase-3 (Abcam, ab2302, 1:500) and LAMP-1 (DHSB H4A3, 1:500) overnight at 4 °C, diluted in PBS containing 1% normal goat serum.

Techniques: Immunocytochemistry, Translocation Assay