anti-pho- nf-κb p65 monoclonal antibody  (Cell Signaling Technology Inc)

Journal: Biology

Article Title: Folate Attenuates Ulcerative Colitis via PI3K/AKT/NF-κB/MLCK Axis Inhibition to Restore Intestinal Barrier Integrity

doi: 10.3390/biology14111573

Figure Lengend Snippet: Colonic transcriptomic analysis after folate administration. ( A ) PCA plot. ( B ) Venn diagram. ( C ) Trend analysis of the DEGs. ( D , E ) Volcano plots. ( F ) KEGG analysis of DEGs. ( G ) Heatmap of DEGs associated with the PI3K/AKT pathway. ( H ) GSEA plots of the PI3K/AKT and NF-κB pathways.

Article Snippet: Primary antibodies used are as follows: GAPDH (1:20,000, Abcam, Cambridge, UK), Occludin (1:2000, Proteintech, Wuhan, China), ZO-1 (1:5000, Proteintech), Claudin-1 (1:1000, Proteintech), Claudin-4 (1:1000, Abcam), E-cadherin (1:2000, CellSignalingTechnology, Danvers, MA, USA), β-catenin (1:1000, CellSignalingTechnology), PI3K (1:5000, Zenbio, Chengdu, China), phos-PI3K (1:1000, CellSignalingTechnology), ERK (1:2000, CellSignalingTechnology), phos-ERK (1:2000, CellSignalingTechnology), AKT (1:2000, Proteintech), NF-κB p65 (1:1000, Proteintech), phos-NF-κB p65 (1:1000, CellSignalingTechnology), MLCK (1:1000, Proteintech), MLC2 (1:1000, Proteintech), phos-MLC2 (1:1000, CellSignalingTechnology).

Techniques:

Journal: Biology

Article Title: Folate Attenuates Ulcerative Colitis via PI3K/AKT/NF-κB/MLCK Axis Inhibition to Restore Intestinal Barrier Integrity

doi: 10.3390/biology14111573

Figure Lengend Snippet: Folate inhibits the PI3K/AKT/NF-κB/MLCK/MLC2 axis. ( A , B ) Immunoblotting analysis of colonic PI3K, AKT, and NF-κB p65 levels and their phosphorylation states ( n = 3). ( C , D ) Immunoblotting analysis of colonic MLCK, MLC2 and phos-MLC2 ( n = 3). ( E , F ) Immunoblotting analysis of cellular levels of PI3K, AKT, and NF-κB p65 and their phosphorylation states ( n = 3). ( G , H ) Immunoblotting analysis of cellular MLCK, MLC2 and phos-MLC2 ( n = 3). p values are provided in the figures and were determined by one-way ANOVA.

Article Snippet: Primary antibodies used are as follows: GAPDH (1:20,000, Abcam, Cambridge, UK), Occludin (1:2000, Proteintech, Wuhan, China), ZO-1 (1:5000, Proteintech), Claudin-1 (1:1000, Proteintech), Claudin-4 (1:1000, Abcam), E-cadherin (1:2000, CellSignalingTechnology, Danvers, MA, USA), β-catenin (1:1000, CellSignalingTechnology), PI3K (1:5000, Zenbio, Chengdu, China), phos-PI3K (1:1000, CellSignalingTechnology), ERK (1:2000, CellSignalingTechnology), phos-ERK (1:2000, CellSignalingTechnology), AKT (1:2000, Proteintech), NF-κB p65 (1:1000, Proteintech), phos-NF-κB p65 (1:1000, CellSignalingTechnology), MLCK (1:1000, Proteintech), MLC2 (1:1000, Proteintech), phos-MLC2 (1:1000, CellSignalingTechnology).

Techniques: Western Blot, Phospho-proteomics

Journal: Chinese Medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Primary antibody sources and dilutions

Article Snippet: Anti-Pho- NF-κB P65 monoclonal antibody , CST , 1:1000.

Techniques:

Journal: Chinese Medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Taraxerone ameliorates sepsis-induced acute lung injury in mice. Taraxerone (0, 1, 6, and 30 mg/kg) was administered intraperitoneally, and the lung tissues and BALFs were collected 12 h after the CLP model was established. A H&E staining was used to detect histopathological changes in lung tissue. The bar represented 100 μm. B The W/D ratio of lung tissue was measured to determine pneumonedema. C BALFs were collected for total protein content and D count of neutrophils. E The data of breathing frequency and dynamic compliance were detected and collected by BUXCO system. F TNF-α, IL-1β, and IL-6 in BALFs were detected by ELISA. G MPO activity in lung tissue. H The Pho-P65, P65, Pho-IκB, and IκB levels in lung tissues were determined using western blotting. I Schematic diagram of the process of screening M1 macrophages by flow cytometry. J – K The change in M1 macrophage proportion was detected using flow cytometry. Data are presented as mean ± SD (n ≥ 6 in each group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Anti-Pho- NF-κB P65 monoclonal antibody , CST , 1:1000.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Flow Cytometry

Journal: Chinese Medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Taraxerone alleviates LPS-induced inflammatory responses in primary macrophages. A Primary macrophages were exposed to different concentrations of taraxerone (0, 1, 3, 10, 30, and 50 μM) for 12 h, and cell viability was detected with CCK8 kit. Primary macrophages were exposed to different concentrations of taraxerone (10, 30, 50 μM) and LPS (1 μg/mL) for 12 h. B mRNA levels of inflammatory cytokines (IL-1β, IL-6, TNF-α, iNOS, and IL-12) in primary macrophages were detected 12 h later. C P65 nuclear translocation was discovered using immunofluorescence staining. The bar represented 20 μm. D Western blotting was used to determine the location of P65 in the nuclei and cytoplasm. E Western blotting detected Pho-P65, P65, Pho-IκB, and IκB protein levels in primary macrophages. F The M1 macrophage proportion was detected using flow cytometry. Data are presented as mean ± SD (n = 3 in each group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Anti-Pho- NF-κB P65 monoclonal antibody , CST , 1:1000.

Techniques: Translocation Assay, Immunofluorescence, Staining, Western Blot, Flow Cytometry

Journal: Chinese Medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: The anti-inflammatory and anti-oxidative stress effects of taraxerone in primary macrophages are exerted by regulating SIRT1. A Mice were pretreated with taraxerone (1, 6, and 30 mg/kg) or saline for 2 h before cecal ligation and puncture. Primary macrophages were treated with taraxerone (10, 30, and 50 μM) and LPS (1 μg/mL). The protein levels of Ace-P65 were detected using western blotting. B The interaction between SIRT1 and P65 was analyzed using CO-IP. C Western blotting was preformed to determine the effects of taraxerone (50 μM) and EX527 (5 μM/mL) on NLRP3, Cleaved Caspase-1, Pro Caspase-1, ASC, Pho-P65, Ace-P65, P65, Pho-IκB, and IκB protein levels. Primary macrophages were pretreated with EX527 (5 μM/mL) for 2 h, and then treated with LPS (1 μg/mL) and taraxerone (50 μM) for 12 h. D Detection of NLRP3, Pro Caspase-1, ASC, and IL-18 mRNA levels. E The activity of Caspase-1 was measured. F The proportion of CD80 + macrophages was detected using flow cytometry. G , H H2DCFDA probe was used to detect ROS levels. The bar represented 100 μm. I Detection of IL-6, IL-1β, TNF-α, iNOS, and IL-12 mRNA levels. J Detailed view of protein-tar interaction. The SIRT1 residues involved in the interaction are represented by a stick model, where hydrogen bonds are represented by dashed lines. Taraxerone is also represented by a stick model. Data are presented as mean ± SD (n = 6 in vivo, n = 3 in vitro). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Anti-Pho- NF-κB P65 monoclonal antibody , CST , 1:1000.

Techniques: Saline, Ligation, Western Blot, Co-Immunoprecipitation Assay, Activity Assay, Flow Cytometry, In Vivo, In Vitro

Journal: Chinese Medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Taraxerone plays a protective role in sepsis-induced ALI mice through the SIRT1 pathway. The EX527 (10 mg/kg) was administered intraperitoneally 2 h before the taraxerone (0, 30 mg/kg) treatment, and the lung tissues and BALFs were collected after the CLP model was constructed 12 h later. A With H&E staining, we examined the lung tissue for any pathological changes. The bar represented 100 μm. B The breathing frequency and dynamic compliance of mice were detected by BUXCO system. C Detection of IL-1β, IL-6, TNF-α, and IL-18 mRNA levels. D ROS level in BALFs were determined using flow cytometry and analyzed with FlowJo. E Changes in the proportion of M1 macrophages in BALFs were detected using flow cytometry, and the data were analyzed using FlowJo. F Effects of taraxerone (30 mg/kg) and EX527 (10 mg/kg) on NLRP3, Cleaved Caspase-1, Pro Caspase-1, ASC, Pho-P65, Ace-P65, P65, Pho-IκB, and IκB protein levels. G – I MDA content, GSH content, and SOD activity were determined to reflect the oxidative stress levels of lung tissues. Data are presented as mean ± SD (n ≥ 6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: Anti-Pho- NF-κB P65 monoclonal antibody , CST , 1:1000.

Techniques: Construct, Staining, Flow Cytometry, Activity Assay

Journal: Chinese Medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Taraxerone plays a protective role in acute lung injury by activating SIRT1. Taraxerone activates SIRT1 to block the NF-κB-NLRP3 inflammasome axis and to suppress the excessive ROS, inhibiting the M1 polarization of macrophages, and exerting a protective effect on acute lung injury mice.

Article Snippet: Anti-Pho- NF-κB P65 monoclonal antibody , CST , 1:1000.

Techniques: Blocking Assay

Journal: Pharmaceuticals

Article Title: Exploring the Mechanism of Asiatic Acid against Atherosclerosis Based on Molecular Docking, Molecular Dynamics, and Experimental Verification

doi: 10.3390/ph17070969

Figure Lengend Snippet: Effects of AA on NF-κB activation and PPARγ expression: ( A ) Western blot results, ( B ) NF-κB p65, p-p65 protein expression levels, and ( C ) PPARγ protein expression levels. The results are presented as mean values ± standard error of the mean (SEM), with statistical significance indicated at a p -value below 0.05 (compared with the control group, # p < 0.05, ## p < 0.01; compared with LPS group, * p < 0.05).

Article Snippet: Primary antibodies against IL6, iNOS, TNF α, COX-2, PPARγ, pho-NF-κB p65, NF-κB p65, and GAPDH were purchased from Proteintech Group, Inc., Wuhan, China.

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Pharmaceuticals

Article Title: Exploring the Mechanism of Asiatic Acid against Atherosclerosis Based on Molecular Docking, Molecular Dynamics, and Experimental Verification

doi: 10.3390/ph17070969

Figure Lengend Snippet: AA regulates macrophage inflammation through the PPARγ/NF-κB signaling pathway: ( A ) Western blot results, ( B ) NF-κB p65, p-p65 protein expression levels, and ( C ) PPARγ protein expression levels. The results are presented as mean values ± standard error of the mean (SEM), with statistical significance indicated at a p -value below 0.05 (** p < 0.01).

Article Snippet: Primary antibodies against IL6, iNOS, TNF α, COX-2, PPARγ, pho-NF-κB p65, NF-κB p65, and GAPDH were purchased from Proteintech Group, Inc., Wuhan, China.

Techniques: Western Blot, Expressing

Journal: RSC Advances

Article Title: Anti-inflammatory constituents isolated from the flowers of Hosta plantaginea via suppression of the NF-κB signaling pathway in LPS-stimulated RAW 264.7 macrophages †

doi: 10.1039/d2ra07623c

Figure Lengend Snippet: Effects of compounds 1 and 3 on NF-κB p65 (A) and phosphorylated NF-κB p65 (B) in LPS-stimulated RAW 264.7 macrophages. Data were presented as the mean ± SD of three experiments. ## p < 0.01 vs. the control group; ** p < 0.01 vs. the LPS only model group.

Article Snippet: Anti-phos-NF-κB p65 (Ser536) and anti-NF-κB p65 antibodies were purchased from Cell Signaling Technology (Boston, USA).

Techniques: Control

Journal: RSC Advances

Article Title: Anti-inflammatory constituents isolated from the flowers of Hosta plantaginea via suppression of the NF-κB signaling pathway in LPS-stimulated RAW 264.7 macrophages †

doi: 10.1039/d2ra07623c

Figure Lengend Snippet: Effects of compounds 1 and 3 on NF-κB p65 (A) and phosphorylated NF-κB p65 (B) in LPS-stimulated RAW 264.7 macrophages. Data were presented as the mean ± SD of three experiments. ## p < 0.01 vs. the control group; ** p < 0.01 vs. the LPS only model group.

Article Snippet: Anti-phos-NF-κB p65 (Ser536) and anti-NF-κB p65 antibodies were purchased from Cell Signaling Technology (Boston, USA).

Techniques: Control