Journal: Experimental and Therapeutic Medicine

Article Title: LncRNA NEAT1 promotes apoptosis and inflammation in LPS-induced sepsis models by targeting miR-590-3p

doi: 10.3892/etm.2020.9079

Figure Lengend Snippet: Inhibition of NEAT1 suppresses the NF-κB signaling pathway via targeting miR-590-3p. H9c2 cells were transfected with si-NEAT1 and anti-miR-590-3p or their corresponding negative controls (si-NC and anti-NC), followed by treatment with LPS. (A) The protein levels of NF-κB pathway-related factors p-p65 and p65 were detected and (B) quantified using western blot analysis. * P<0.05. NEAT1, nuclear enriched abundant transcript 1; miR, microRNA; si-NEAT1, small interfering RNA targeting NEAT1; LPS, lipopolysaccharide; p, phosphorylated.

Article Snippet: Primary antibodies against Bcl-2 (1:500; cat. no. BA0412), Bax (1:1,000; cat. no. BA0315-2), caspase 3 (1:1,000; cat. no. BA3257), TNF receptor associated factor 6 (TRAF6; 1:1,500; cat. no. A00185), phosphorylated (p)-p65 (1:1,000; cat. no. P00284), p65 (1:2,000; cat. no. A00284) and GAPDH (1:2,000; cat. no. BA2913) were purchased from Wuhan Boster Biological Technology, Ltd. Membranes were then incubated with horseradish peroxidase-labeled secondary antibody (1:5,000; cat no. BA1056; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature.

Techniques: Inhibition, Transfection, Western Blot, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: JNK-IN-8 treatment alleviates lipopolysaccharide-induced acute lung injury via suppression of inflammation and oxidative stress regulated by JNK/NF-κB signaling

doi: 10.3892/mmr.2020.11789

Figure Lengend Snippet: JNK-IN-8 suppresses the JNK/NF-κB signaling pathway in LPS-induced acute lung injury in vivo and in vitro . (A) Primary macrophages stimulated by LPS and JNK-IN-8 (10 mM) for 2 h were extracted and subjected to western blot analysis. The protein expression levels of JNK, p-JNK, p65 and p-p65 were assessed and (B) analyzed by densitometry. (C) Primary macrophages, stimulated with LPS in the presence or absence of JNK-IN-8 (10 mM) for 0, 30, 60 or 120 min, were extracted and subjected to western blot analysis. The protein expression levels of JNK, p-JNK, p65 and p-p65 were assessed and (D) analyzed by densitometry. (E) Protein expression levels of JNK, p-JNK, p65 and p-p65 in lung tissue were assessed and (F) analyzed by densitometry. β-actin was used as the internal loading control. (G) Primary macrophages were stimulated with LPS in the presence or absence of JNK-IN-8 (10 mM) for 2 h. Cytoplasmic and nuclear NF-κB p65 protein expression levels were determined and (H) analyzed by densitometry. β-actin and Lamin B were used as internal loading controls. Data are presented as the mean ± SD (n=3). ***P<0.001 vs. Sham; ## P<0.01 and ### P<0.001 vs. LPS. LPS, lipopolysaccharide; p-, phosphorylated.

Article Snippet: Specific primary antibodies against NF-κB p65 (cat. no. 8242), phosphorylated (p)-p65 (Ser536; cat. no. 3303), JNK (cat. no. 9252), p-JNK (Thr183/Tyr185; cat. no. 4668) and β-actin (cat. no. 3700) were purchased from Cell Signaling Technology, Inc.

Techniques: In Vivo, In Vitro, Western Blot, Expressing, Control

Journal: Mediators of Inflammation

Article Title: Interference in Macrophage Balance (M1/M2): The Mechanism of Action Responsible for the Anti-Inflammatory Effect of a Fluorophenyl-Substituted Imidazole

doi: 10.1155/2024/9528976

Figure Lengend Snippet: Effect of fluorophenyl-imidazole on TLR-4 receptor (CD284-MD2) (a); mannose receptor (CD206 hi+ ) (b); phosphorylated protein p65 (c); apoptosis (d); and phagocytic activity (e) in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL). The experimental groups used were B: cells pretreated with vehicle and incubated with PBS (blank group); LPS: cells pretreated with vehicle and stimulated with LPS (1 μ g/mL); dexa + LPS: cells pretreated with dexamethasone (7 μ M) and stimulated with LPS (1 μ g/mL); flu (1 μ M): cells pretreated with the best concentration (based on IC 50 values) (1 µ M) of fluorophenyl-imidazole alone; and (flu 1 μ M) + LPS: fluorophenyl-imidazole (1 µ M), before LPS (1 μ g/mL). Taxel (30 μ M) was used alone in apoptosis experiments, as positive control of apoptosis. The results for TLR-4 (CD284-MD2) and (CD206 hi+ ) were quantified as receptor expression percentage in relation to cells stained with control isotype antibody while, phosphorylated protein p65 expression was compared to the blank control (cells with no treatment) that represents the basal expression phosphorylated p65 protein. The apoptosis values (%) were quantified as the percentage of apoptotic cells compared with the total cell number and the phagocytic index was measured using the following equation: phagocytic index (PI) = A 1/ A 0; where A 1 is the absorbance of sample, LPS and dexamethasone, and A 0 is the absorbance of black control. The results were expressed as the mean ± SEM; n = 3; ns, not significant; ∗∗ P < 0.01; and ∗∗∗ P < 0.001.

Article Snippet: Afterward, they were transferred to ELISA microplates containing monoclonal antibodies specific against the phosphorylated p 65 protein (PathScan®Phospho-NF- κ B p65 (Ser536) ELISA Kit (Cell Signalling Technology, Inc., Danvers, Massachusetts, USA)).

Techniques: Activity Assay, Incubation, Concentration Assay, Positive Control, Expressing, Staining, Control

Journal: Frontiers in cell and developmental biology

Article Title: Mechanical Stimulation Protects Against Chondrocyte Pyroptosis Through Irisin-Induced Suppression of PI3K/Akt/NF-κB Signal Pathway in Osteoarthritis.

doi: 10.3389/fcell.2022.797855

Figure Lengend Snippet: FIGURE 5 | Expression of inflammation-related markers and immunofluorescence analysis of NF-κB p65 in chondrocytes. (A–D) The relative protein expressions were detected in the control (CG), IL-1β group, IL-1β + irisin (5 ng/ml), and IL-1β + irisin (10 ng/ml) groups. Chondrocytes were preincubated with different concentrations of irisin (0, 5, or 10 ng/ml) and treated with IL-1β (10 ng/ml) for 24 h. The levels of expression of ADAMTS-5 and MMP-13 were increased after treatment with IL-1β, whereas decreased after treatment with irisin. In addition, irisin-treated chondrocytes could partially recover the expression of chondrocyte-specific collagen II. The phosphorylation of PI3K, Akt, and NF-κB p65 was significantly suppressed in the irisin-treated group compared with that in the IL-1β group. (E) Relative mRNA (Continued)

Article Snippet: Antibodies used included anti-FNDC5 antibody (ab174833, 1:1,500, Abcam), anti-collagen II antibody (ab188570, 1:2,000, Abcam), anti-MMP-13 antibody (ab39012, 1:2,000, Abcam), anti-ADAMTS-5 antibody (ab41037, 1:2,000, Abcam), anti-PI3K antibody (ab32089, 1:2,000, Abcam), phosphorylated (p)-PI3 kinase antibody (4228T, 1:2,000, Cell Signaling Technology), antiAkt antibody (abs131788, 1:2,000, Absin Bioscience Inc.), phosphorylated (p)-Akt antibody (4058L, 1:2,000, Cell Signaling Technology), anti- NF-kappaB p65 antibody (ab16502, 1:2,000, Abcam), phosphorylated (p)-NF-kappaB p65 antibody (S536, 1:2,000, Cell Signaling Technology), NLRP3 polyclonal antibody (19771-1-AP, 1:2,000, Proteintech Group, Rosemont, IL, United States), caspase 1/p20/p10 polyclonal antibody (22915-1-AP, 1:2,000, Proteintech Group), and anti-β-actin antibody (205361-AP, 1:3,000, Proteintech Group).

Techniques: Expressing, Control, Phospho-proteomics

Journal: Cancer Science

Article Title: DEAD ‐box helicase 1 inhibited CD8 + T cell antitumor activity by inducing PD‐L1 expression in hepatocellular carcinoma

doi: 10.1111/cas.16076

Figure Lengend Snippet: DDX1 expression correlated positively with PD‐L1 expression in HCC patients. (A) Heat map revealing the correlation between DDX1 and immune checkpoints (TIGIT, PDCD1, HAVCR2, CTLA4, CD274) in different cancers. (B) Scatter diagram showing the correlation between DDX1 and PD‐L1 in patients with HCC. (C) Multicolor fluorescence demonstrating the localization relationship between DDX1 and PD‐L1 expression. (D) GEO database (LIHC GSE125449) analysis for determining CD274 (PD‐L1), DDX1, IFNG, RELA (p65) and RELB expression between patients treated with or without antibody against PD‐L1/CTLA4. * p < 0.05; ** p < 0.01; *** p < 0.001; N.S., not significant.

Article Snippet: The primary antibodies were raised against DDX1 (1:1000; Proteintech), PD‐L1 (1:500; Proteintech), phosphorylated (p)‐p65 (1:1000; ZENBIO), and p‐65 (1:1000; ZENBIO).

Techniques: Expressing, Fluorescence

Journal: Cancer Science

Article Title: DEAD ‐box helicase 1 inhibited CD8 + T cell antitumor activity by inducing PD‐L1 expression in hepatocellular carcinoma

doi: 10.1111/cas.16076

Figure Lengend Snippet: DDX1 expression enhanced IFN‐γ‐induced PD‐L1 expression on tumor cells via p65 phosphorylation. (A) DDX1 mRNA (upper panel) and protein expression (lower panel) levels in the LO2 normal human hepatic cell line and in liver cancer cell lines (Huh7, SMMC‐7721, QGY‐7703, HepG2). (B–D) Western blot analysis of DDX1, PD‐L1, p‐p65, and p65 in Huh7 and HepG2 cells transfected with DDX1 overexpression plasmid (B) and under IFN‐γ stimulation (C, D). (E) DDX1 and RelA localization was determined by multicolor fluorescence with anti‐DDX1 antibody (green), anti‐p65 antibody (red), and the nucleus (blue). (F) Co‐IP of DDX1 and p65. HepG2 cells were treated with or without IFN‐γ (100 ng/mL) for 1 h. The lysates were immunoprecipitated with DDX1 antibody or control IgG. Immunoblotting assay was performed with p65 antibody. * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were raised against DDX1 (1:1000; Proteintech), PD‐L1 (1:500; Proteintech), phosphorylated (p)‐p65 (1:1000; ZENBIO), and p‐65 (1:1000; ZENBIO).

Techniques: Expressing, Phospho-proteomics, Western Blot, Transfection, Over Expression, Plasmid Preparation, Fluorescence, Co-Immunoprecipitation Assay, Immunoprecipitation, Control

Journal: International Journal of Molecular Medicine

Article Title: Suppressive effect mediated by human adipose-derived stem cells on T cells involves the activation of JNK

doi: 10.3892/ijmm.2018.3953

Figure Lengend Snippet: Analysis of signaling pathways. (A) Protein levels of NF-κB P65, p-P65, IKKβ and IκBα in the total cell fraction of control and co-cultured Jurkat cells and (B) densitometric analysis of the western blot data. (C) Expression of NF-κB P65 and p-P65 in the nuclear protein of Jurkat cells and (D) densitometric analysis of the western blot data. Levels of NF-κB p-P65 were decreased in the total cell and nuclear fractions of co-cultured Jurkat cells at 72 h. NF-κB, nuclear factor-κB; p-, phosphorylated; IκBα, inhibitor of NF-κBα; IKK, IκB kinase β; J, control; co, co-cultured.

Article Snippet: The membranes were probed with primary antibodies against phosphorylated (p)-P65 (1:2,000, cat. no. AF2006, Affinity Biosciences, Cambridge, UK), P65 (1:2,000, cat. no. 10745-1-AP, ProteinTech Group, Inc., Wuhan, China), inhibitor of NF-κB (IκB) kinase (IKK)β (1:1,000, cat. no. Ab124957, Abcam, Cambridge, MA, USA), IκBα (1:1,000, cat. no. 4812, Cell Signaling Technology, Inc., Danvers, MA, USA), B-cell lymphoma 2 (Bcl-2; 1:2,000, cat. no. 12789-1-AP, ProteinTech Group, Inc.), Bcl-2-associated X protein (Bax; 1:5,000, cat. no. 50599-2-IG, Protein Tech Group, Inc), JNK1/2 (1:1,000, cat. no. 9252, Cell Signaling Technology, Inc.), p-JNK1/2 (1:1,000, cat. no. 4668, Cell Signaling Technology, Inc.), extracellular signal-regulated kinase (ERK)1/2 (1:1,000, cat. no. 9102, Cell Signaling Technology, Inc.), p-ERK1/2 (1:2,000, cat. no. 4370, Cell Signaling Technology, Inc.), P38 (1:1,000, cat. no. 9212, Cell Signaling, Technology, Inc.), p-P38 (1:1,000, cat. no. 9211, Cell Signaling Technology, Inc.), mothers against decapentaplegic (Smad)2/3 (1:1,000, cat. no. AF6367, Affinity Biosciences), p-Smad2/3 (1:200, cat. no. MAB8935, R&D Systems, Inc.) and the loading control β-actin (1:200, cat. no. BM0627, Boster Biological Technology, Wuhan, China).

Techniques: Protein-Protein interactions, Control, Cell Culture, Western Blot, Expressing

Journal: Arhiv za bioloske nauke

Article Title: Contribution of O-GlcNAc modification of NF-κB p65 in the attenuation of diabetes-induced haptoglobin expression in rat liver

doi: 10.2298/abs200928049m

Figure Lengend Snippet: Fig. 2. Diabetes-related changes in nuclear abundance and binding activities of NF-κB p65, GR and STAT3 for the hormone responsive-element (HRE) of the Hp gene promoter. A – Nucleotide sequence of the cis-acting element B (-88/- 146 bp) from the HRE (-165/-56 bp) of the rat Hp gene containing the IL-6 and glucocorticoid-responsive element. Overlapping binding sites for STAT3 and GR are framed. B – Immunoblot analysis of liver nuclear extracts (NE) from ND and D rats with anti-NF-κB p65, anti-GR and anti-STAT3 antibodies. C – Immunoblot analysis of nuclear proteins (NP) from ND and D rats after DNA-affinity chro- matography. Immunoblots obtained from at least three independent experiments were quantified using densitometric analysis and are presented in the graphs. The data are expressed as fold-increases with respect to ND rats. ND – control rats; D2, D4, D8 – 2nd, 4th and 8th week after STZ treatment. The values in the graphs are presented as the means±SEM. a,b,c,dMean values with unlike letters were significantly different (P<0.05).

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Blotto Base buffer (0.1% Tween 20, 20 mM Tris–HCl, pH 7.6, 137 mM NaCl) and incubated for an additional 2 h at room temperature in the same buffer containing rabbit monoclonal anti-Hp antibody (Abcam, Cambridge, UK), rabbit polyclonal antibodies specific to NF-κB p65, phosphorylated (p) NF-κB p65, STAT3, p-Tyr STAT3, p-Ser STAT3 and GR (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Binding Assay, Sequencing, Western Blot, Control

Journal: Arhiv za bioloske nauke

Article Title: Contribution of O-GlcNAc modification of NF-κB p65 in the attenuation of diabetes-induced haptoglobin expression in rat liver

doi: 10.2298/abs200928049m

Figure Lengend Snippet: Fig. 3. Coimmunoprecipitation analysis of changes in interaction between STAT3, NF-κB p65 and GR in the liver nuclear extracts during diabetes. Representative immunoblots of immunoprecipi- tated GR (IP:GR) (A), STAT3 (IP:STAT3) (B), and NF-κB p65 (IP:NF-κB p65) (C) from liver nuclear extracts (NE) of ND and D rats with anti-GR, anti-STAT3 and anti-NF-κB p65 antibodies. ND – control rats; D2, D4, D8 – 2nd, 4th and 8th week after STZ treatment, respectively.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Blotto Base buffer (0.1% Tween 20, 20 mM Tris–HCl, pH 7.6, 137 mM NaCl) and incubated for an additional 2 h at room temperature in the same buffer containing rabbit monoclonal anti-Hp antibody (Abcam, Cambridge, UK), rabbit polyclonal antibodies specific to NF-κB p65, phosphorylated (p) NF-κB p65, STAT3, p-Tyr STAT3, p-Ser STAT3 and GR (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Control

Journal: Arhiv za bioloske nauke

Article Title: Contribution of O-GlcNAc modification of NF-κB p65 in the attenuation of diabetes-induced haptoglobin expression in rat liver

doi: 10.2298/abs200928049m

Figure Lengend Snippet: Fig. 4. Diabetes-related changes in O‑GlcNAcylation and phosphorylation of nuclear NF-κB p65 and STAT3 proteins. A – Electrophoretic profile of liver nuclear proteins (NP) from ND and D rats after elution from the wheat germ agglutinin (WGA) column. B – Immunoblot analysis with anti-NF-κB p65 antibody of O-GlcNAc-modified liver nuclear proteins (NP) eluted from the WGA column. C – Immunoblot analysis with anti-phosphorylated (p)-NF-κB p65, anti-p- STAT3 (Tyr) and anti-p- STAT3 (Ser) antibodies of liver nuclear extracts. Representative gel and blots from three independent experiments are shown. D – Immunoblots obtained from at least three independent experiments were quantified using densitometric analysis and are presented in the graphs. The data are expressed as fold increase with respect to ND rats. ND – control rats; D2, D4, D8 – 2nd, 4th and 8th weeks after STZ treatment. The values in the graphs are presented as the means±SEM. a,b,cMean values with unlike letters were significantly different (P<0.05).

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Blotto Base buffer (0.1% Tween 20, 20 mM Tris–HCl, pH 7.6, 137 mM NaCl) and incubated for an additional 2 h at room temperature in the same buffer containing rabbit monoclonal anti-Hp antibody (Abcam, Cambridge, UK), rabbit polyclonal antibodies specific to NF-κB p65, phosphorylated (p) NF-κB p65, STAT3, p-Tyr STAT3, p-Ser STAT3 and GR (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Modification, Control

Journal: Glia

Article Title: IL-27 Inhibits OSM-mediated TNF-? and iNOS Gene Expression in Microglia

doi: 10.1002/glia.20989

Figure Lengend Snippet: IL-27 inhibits OSM-mediated p65 activation and recruitment to the TNF-α and iNOS promoters. A. BV2 microglial cells were treated with media alone, OSM (10 ng/ml) for 0.25 h, IL-27 (5 ng/ml) for 4.25 h, or pre-treated with IL-27 for 4 h and then treated with OSM for 0.25 h. Protein lysates were collected and analyzed for P-p65Ser536 and total p65 by immunoblotting. Representative of 2 independent experiments. B. BV2 cells were treated with media alone, OSM (10 ng/ml) for 2 h, IL-27 (5 ng/ml) for 6 h, or pre-treated with IL-27 for 4 h and then treated with OSM for 2 h. The ChIP assay was performed to assess binding of P-p65 Ser536, total p65, AcH3, p300 and HDAC1 to NF-κB sites within the TNF-α and iNOS promoters. Total IgG shows no non-specific binding. Input serves as a control for total DNA levels.

Article Snippet: Abs against phosphorylated (p)-p65 Ser276 , p-p65 Ser536 , total p65, p-STAT1 Tyr701 , p-STAT3 Tyr705 , total STAT3, p-p38, p-p44/42 Thr202/Tyr204 and p-Akt were obtained from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Activation Assay, Western Blot, Binding Assay