nf-κb (p65) transcription factor assay kit  (Cayman Chemical)

Journal: Journal of Inflammation Research

Article Title: Astragaloside IV Attenuates Angiotensin II-Induced Inflammatory Responses in Endothelial Cells: Involvement of Mitochondria

doi: 10.2147/JIR.S504427

Figure Lengend Snippet: Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced monocyte adhesion to endothelial cells ( A and B ) and the activation of NF-κB ( C ) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h or 30 min. Subsequently, HUVECs were coincubated with fluorescence-labeled human monocytes for 30 min or the NF-κB activity in HUVECs was assayed by the use of a nonradioactive ELISA-based assay kit. ( A ) Fluorescent images of monocyte adhesion to HUVECs were visualized with a fluorescence microscope at 485/535 nm. Magnification, × 200. HUVECs were incubated with (a) a vehicle (control), (b) Ang II alone, Ang II with AS-IV at the concentration of (c) 3, (d) 10, or (e) 30 µM before coincubation with fluorescence-labeled monocytes. ( B ) Fluorescence intensity of monocyte attachment to HUVECs was quantified using a spectrofluorometer. ( C ) The activities of NF-κB in HUVECs were assayed using a specific TransAM NF-κB p65 transcription factor assay kit. Results are expressed as means ± SE. * p < 0.05 compared with the control group; # p < 0.05 and ## p < 0.01 compared with HUVECs exposed to Ang II alone.

Article Snippet: The specific TransAM NF-κB p65 transcription factor assay kit and nuclear extraction kit were supplied by Active Motif (Carlsbad, CA, USA).

Techniques: Activation Assay, Incubation, Fluorescence, Labeling, Activity Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Control, Concentration Assay, Transcription Factor Assay

Journal: Future Science OA

Article Title: Resveratrol alleviates depressive-like behavior via the activation of SIRT1/NF-κB signaling pathway in microglia

doi: 10.1080/20565623.2025.2463852

Figure Lengend Snippet: Resveratrol treatment suppressed the chronic unpredictable mild stress-induced changes of the protein levels of Sirt1, NF-κB p65, ac-p65, and Iκ-Bα in the mouse hippocampus via Western blot. *P < 0.05 vs. the control group; & P < 0.05 vs. the depression model group.

Article Snippet: NF-κB p65 DNA binding activity was detected using Trans AM NF-κB p65 Chemi Tran-scription Factor Assay Kit (Active Motif, Carlsbad, CA) according to the instructions of manufacturer.

Techniques: Western Blot, Control

Journal: Future Science OA

Article Title: Resveratrol alleviates depressive-like behavior via the activation of SIRT1/NF-κB signaling pathway in microglia

doi: 10.1080/20565623.2025.2463852

Figure Lengend Snippet: Resveratrol treatment suppressed the chronic unpredictable mild stress-induced changes of the protein levels of Sirt1, NF-κB p65, ac-p65, and Iκ-Bα in the mouse prefrontal cortex via Western blot. *P < 0.05 vs. the control group; & P < 0.05 vs. the depression model group.

Article Snippet: NF-κB p65 DNA binding activity was detected using Trans AM NF-κB p65 Chemi Tran-scription Factor Assay Kit (Active Motif, Carlsbad, CA) according to the instructions of manufacturer.

Techniques: Western Blot, Control

Journal: Future Science OA

Article Title: Resveratrol alleviates depressive-like behavior via the activation of SIRT1/NF-κB signaling pathway in microglia

doi: 10.1080/20565623.2025.2463852

Figure Lengend Snippet: Resveratrol treatment suppressed the LPS-induced changes of the protein levels of Sirt1, NF-κB p65, ac-p65, and Iκ-Bα in BV2 cells via Western blot. *P < 0.05 vs. the control group; **P < 0.01 vs. the control group; ***P < 0.001 vs. the control group.

Article Snippet: NF-κB p65 DNA binding activity was detected using Trans AM NF-κB p65 Chemi Tran-scription Factor Assay Kit (Active Motif, Carlsbad, CA) according to the instructions of manufacturer.

Techniques: Western Blot, Control

Journal: Heliyon

Article Title: Acacetin protects against acute lung injury by upregulating SIRT1/ NF-κB pathway

doi: 10.1016/j.heliyon.2024.e37083

Figure Lengend Snippet: Acacetin improved the function of SIRT1 and inhibited the acetylated activation of NF-κB-p65. The levels of NAD + (A), the ratio of NAD + /NADH (B), the mRNA expression of SIRT1 (C), and the activity of SIRT1 (D) were detected in the lung tissue from LPS-treated mice with or without acacetin administration. The representative immunoblots of SIRT1, acetylated NF-κB-p65, and NF-κB-p65 in the nuclear protein, H3 was used as a loading control (E). The levels of SIRT1 (F), acetylated NF-κB-p65 (G), nuclear NF-κB-p65 (H), the ratio of acetylated NF-κB-p65/nuclear NF-κB-p65 (I) were analyzed ( n = 3 independent experiments). The DNA binding activity of NF-κB (J) were assessed ( n = 5 independent experiments). Data were means ± S. D., n = 10 mice/group. ** P < 0.01 vs. control group, ## P < 0.01 vs. LPS group.

Article Snippet: NF-κB-p65 Transcription Factor Assay Kit (#10007889) was purchased from Cayman Chemical (Michigan, USA).

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Control, Binding Assay

Journal: Heliyon

Article Title: Acacetin protects against acute lung injury by upregulating SIRT1/ NF-κB pathway

doi: 10.1016/j.heliyon.2024.e37083

Figure Lengend Snippet: Acacetin up-regulated SIRT1 function and restrained NF-κB-p65 acetylated activation in A549 cells. A549 cells were treated with TNF-α (10 ng/ml) in the presence or absence of acacetin (0, 0.3, 1, 3 μM) for 24 h. The levels of NAD + (A), the ratio of NAD + /NADH (B), the mRNA expression of SIRT1 (C), and the activity of SIRT1 (D) were detected. The representative immunoblots of SIRT1, acetylated NF-κB-p65, and NF-κB-p65 in the nuclear protein, H3 was used as a loading control (E). The levels of SIRT1 (F), acetylated NF-κB-p65 (G), nuclear NF-κB-p65 (H), the ratio of acetylated NF-κB-p65/nuclear NF-κB-p65 (I) were analyzed ( n = 3 independent experiments). The DNA binding activity of NF-κB (J) were assessed. Data were means ± S. D., n = 5 independent experiments. ** P < 0.01 vs. control group, ## P < 0.01 vs. TNF-α treated group.

Article Snippet: NF-κB-p65 Transcription Factor Assay Kit (#10007889) was purchased from Cayman Chemical (Michigan, USA).

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Control, Binding Assay

Journal: Heliyon

Article Title: Acacetin protects against acute lung injury by upregulating SIRT1/ NF-κB pathway

doi: 10.1016/j.heliyon.2024.e37083

Figure Lengend Snippet: Silencing SIRT1 abolished the efficacy of acacetin for improving SIRT1 function and restraining NF-κB-p65 acetylated activation induced by TNF-α. A549 cells were transfected with Control siRNA or SIRT1 siRNA to further investigate the relationship of acacetin and SIRT1. (A) A549 cells viabilities were measured by MTT assay. (B–E) The contents of LDH, TNF-α, IL-1β, IL-6, and IL-17 in the supernatant of A549 cells were evaluated. (F) The activity of SIRT1 were detected. (G) The representative immunoblots of SIRT1, acetylated NF-κB-p65, and NF-κB-p65 in the nuclear protein, H3 was used as a loading control. The levels of SIRT1 (H), acetylated NF-κB-p65 (I), nuclear NF-κB-p65 (J), and the ratio of acetylated NF-κB-p65/nuclear NF-κB-p65 (K) were analyzed ( n = 3 independent experiments). (L) The DNA binding activity of NF-κB were assessed. Data were means ± S. D., n = 5 independent experiments. ** P < 0.01 vs. control group, ## P < 0.01 vs. TNF-α treated group, $$ P < 0.01 vs. TNF-α+Acacetin group.

Article Snippet: NF-κB-p65 Transcription Factor Assay Kit (#10007889) was purchased from Cayman Chemical (Michigan, USA).

Techniques: Activation Assay, Transfection, Control, MTT Assay, Activity Assay, Western Blot, Binding Assay

Journal: Cell reports

Article Title: Alternative mRNA splicing events and regulators in epidermal differentiation

doi: 10.1016/j.celrep.2024.113814

Figure Lengend Snippet: (A) Sashimi plot showing enriched relative expression of exon-12-containing isoforms of MAP3K7 upon epidermal differentiation. (B) Scheme of RT-PCR primers (red triangles) to discern MAP3K7 isoforms containing (MAP3K7-long) or excluding exon 12 (MAP3K7-short). Purple bars represent isoform-targeting siRNAs for short and long MAP3K7 transcripts. Bottom, RT-PCR of in vitro progenitor (P) and differentiated (D) keratinocytes and in vivo laser capture microdissected skin tissue of basal (Bs) progenitor and suprabasal (Sb) differentiated layers. CCND1 and LOR are control transcripts enriched in progenitor and differentiated states, respectively. (C) RT-PCR evaluating MAP3K7 isoform expression after transfection of isoform-targeting siRNAs against short and long isoforms. RPL32 is an invariant expression control. (D) Immunoblot for proteins in the NF-κB signaling pathway in progenitor keratinocytes after treatment with control or MAP3K7-isoform-targeted siRNAs, in the presence or absence of tumor necrosis factor α (TNF-α; 10 ng/mL), to stimulate NF-κB signaling. TNF-α was applied for 30 min, and protein lysates were harvested 48 h later. (E) Intensity quantitation of phospho-p65/RelA from immunoblot experiments. Phosphorylated p65 was normalized internally to total p65, and the unstimulated siCTRL signal was set to 1 for each biological replicate. Data are means ± SEM (n = 3) (one-way ANOVA with a Tukey’s honestly significant difference [HSD] post hoc test), *p < 0.05. (F) RT-PCR and immunoblot for TAK1, the protein product of MAP3K7, after overexpression (OE) of each isoform. (G) ELISA for phosphorylated p65 in keratinocytes after OE of empty vector or MAP3K7 short/long isoforms. Data are means ± SEM (n = 3) (one-way ANOVA with a Tukey’s HSD post hoc test), *p < 0.05.

Article Snippet: TransAM NF-κB p65 Transcription Factor ELISA Kit , Active Motif , Cat# 40097.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, In Vivo, Control, Transfection, Western Blot, Quantitation Assay, Over Expression, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Cell reports

Article Title: Alternative mRNA splicing events and regulators in epidermal differentiation

doi: 10.1016/j.celrep.2024.113814

Figure Lengend Snippet: (A) Immunoblot for proteins in the NF-κB signaling pathway after treatment with control or MAP3K7-isoform-targeted siRNAs, in the presence of absence of protein kinase C (PKC), to stimulate NF-κB signaling. (B) Immunofluorescence of Ki-67 and KRT10 in epidermal organotypic tissues generated with CTRL, MAP3K7-long, or MAP3K7-short siRNA-treated primary epidermal keratinocytes. Dotted white lines denote the basement membrane. Arrowheads highlight examples of proliferating keratinocytes detected in suprabasal layers. PKC treatment was applied for 2 h on the day prior to endpoint to evaluate the effect of NF-κB activation on the tissue phenotype. Scale bars (gray): 50 μm. (C and D) Quantitation of (C) Ki-67+ cells and (D) KRT10 signal in epidermal organotypic tissues generated with CTRL, MAP3K7-long, or MAP3K7-short siRNAs. Data are means ± SEM (one-way ANOVA with a Tukey’s HSD post hoc test) *p < 0.05. (E) Quantitative RT-PCR of progenitor-associated genes in epidermal organotypic tissues. Relative expression of each gene is shown for each isoform-specific knockdown relative to its expression in control (gray bars, normalized to 1.0). Data are means ± SEM (n = 3). (F) Quantitative RT-PCR of differentiation-associated genes in epidermal organotypic tissues. Data are means ± SEM (n = 3). (G) Working model of MAP3K7 isoform spatial expression within the epidermis and proposed effects of each isoform on NF-κB signaling and epidermal differentiation.

Article Snippet: TransAM NF-κB p65 Transcription Factor ELISA Kit , Active Motif , Cat# 40097.

Techniques: Western Blot, Control, Immunofluorescence, Generated, Membrane, Activation Assay, Quantitation Assay, Quantitative RT-PCR, Expressing, Knockdown

Journal: Cell reports

Article Title: Alternative mRNA splicing events and regulators in epidermal differentiation

doi: 10.1016/j.celrep.2024.113814

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TransAM NF-κB p65 Transcription Factor ELISA Kit , Active Motif , Cat# 40097.

Techniques: Recombinant, Blocking Assay, Staining, Lysis, Transfection, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Alternative mRNA splicing events and regulators in epidermal differentiation

doi: 10.1016/j.celrep.2024.113814

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TransAM NF-κB p65 Transcription Factor ELISA Kit , Active Motif , Cat# 40097.

Techniques: Recombinant, Blocking Assay, Staining, Lysis, Transfection, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Software, Imaging, Real-time Polymerase Chain Reaction