anti- nf-κb (p65) primary antibody #8242  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: Hif1α promoted the nuclear translocation of NF-κB p50 to inhibit Asmt. A-B. qPCR detected the relative mRNA level of Asmt in PMVECs; C .The immunoblotting and quantitative data showed the effect of si-Hif1α on Asmt in PMVECs; D. ELISA measured the effect of si-Hif1α on the level of 5-MTP in the supernatants of PMVECs. E. The motif and possible binding sites of NF-κB p50 in the promoter region of Asmt; F. ChIP-qPCR detected the binding of NF-κB p50 in Asmt; G . Immunofluorescence staining and quantitative data of NF-κB p50 nuclear translocation. Scale bar, 25 μm; H . qPCR detected the effect of PDTC (30 μM, 30min) on the relative mRNA level of Asmt in PMVECs; I. The immunoblotting and quantitative data showed the effect of PDTC (30 μM, 30min) on Asmt in PMVECs; J. ELISA measured the effect of PDTC (30 μM, 30min) on the level of 5-MTP in the supernatants of PMVECs. A-J. Values were mean ± SEM for n = 3; A. ∗ p < 0.05, compared with the Control group; B-D. ∗ p < 0.05, compared with the Normoxia + si-NC group; # p < 0.05, ## p < 0.01, compared with the Hypoxia + si-NC group; F. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, compared with the corresponding IgG group. ### p < 0.001, compared with the corresponding Control group; G-J. ∗∗ p < 0.01, compared with the Control + PBS group; # p < 0.05, ## p < 0.01, compared with the Hypoxia + PBS group. qPCR, quantitative polymerase chain reaction; PMVECs, mouse pulmonary microvascular endothelial cells; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; NC, negative control; PDTC, pyrrolidinedithiocarbamate. Statistical significance was evaluated using t -test for A and one-way ANOVA for B-J.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, ChIP-qPCR, Immunofluorescence, Staining, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control

Journal: Marine drugs

Article Title: Identification of TRPV1-Inhibitory Peptides from Takifugu fasciatus Skin Hydrolysate and Their Skin-Soothing Mechanisms.

doi: 10.3390/md23050196

Figure Lengend Snippet: Figure 5. Effect of T14 on NF-κB translocation in lipopolysaccharide (LPS)-induced keratinocyte (HaCaT) cells. Western blot analysis was performed to assess the impact of T14 treatment on p65 expression in total cellular proteins. HaCaT cells were grown in 6-well plates, pretreated with varying concentrations of T14 for 6 h, and then co-incubated with CAP for 24 h. After treatment, cell lysates were collected and subjected to Western blotting using a p65-specific antibody. Protein band intensities were quantified using ImageJ software to determine changes in p65 expression levels. Data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison tests. Mean values marked with “*” differ significantly from the model group (p < 0.05).

Article Snippet: Primary antibodies against NF-κB p65 (8242T, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (CL594-6004, Abclonal, Wuhan, China) were diluted according to the manufacturer’s guidelines and incubated overnight at 4 ◦C.

Techniques: Translocation Assay, Western Blot, Expressing, Incubation, Software, Comparison

Journal: Journal of Cosmetic Dermatology

Article Title: Platelet‐Released Growth Factors (PRGFs) Activate NRF2‐ARE and Modulate Inflammatory Response in an NRF2‐Dependent Manner in Primary Human Keratinocytes

doi: 10.1111/jocd.70228

Figure Lengend Snippet: NF‐κB response was suppressed by PRGF‐NRF2‐activating treatment. (A) Cells were stimulated and subjected to western blotting using specific (B) NF‐κB‐p65, (C) p‐NF‐κB‐p65 (Ser 536) and α‐tubulin antibodies ( n = 3). (D) NHEK sin‐lenti‐NF‐κB‐Luc cells were pre‐conditioned with PRGF (5%, 10% PRGF) or 10 μM SFN for 6, 12, and 24 h, followed by media exchange and immediate co‐stimulation with 10 ng/mL TNF‐α for another 6 h. The luminescence values of the experimental group (treated with DMSO) were used only as a control and were set to 1. Single stimulation with 10 ng/mL TNF‐α without NRF2 activating pre‐conditioning was used as a negative control ( n = 6). (E) Keratinocytes were stimulated with 10 μM SFN, 10% PRGF, 10 ng/mL TNF‐α and co‐stimulated with 10 ng/mL TNF‐α + 10% PRGF for various time points. Supernatants were subjected to TNF‐α‐ELISA. Data are shown as the mean ± SD; statistical significance is indicated at * p < 0.05 and ** p < 0.005 versus the control group.

Article Snippet: The NQO1 primary antibody (Abcam UK, #80588, 1:1000, diluted in 5% milk TBS‐T), HO‐1 primary antibody (Santa Cruz, #J1112, 1:1000, diluted in 5% milk TBS‐T), NF‐κB p65 primary antibody (Cell Signaling Technology, USA, #4764, 1:1000, diluted in 5% milk TBS‐T), and p‐NF‐κB‐p65 (Ser 536) (Cell Signaling Technology, USA, #3031, 1:1000, diluted in 5% milk TBS‐T) primary antibody were incubated for 1 h at room temperature.

Techniques: Western Blot, Control, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Anti-Inflammatory Properties of Yellow Passion Fruit Bagasse Extract and Its Potential Role in Periodontal Wound Healing In Vitro

doi: 10.3390/biomedicines13051134

Figure Lengend Snippet: Figure 4. Immunofluorescence analysis of NF-κB nuclear translocation in PDL cells exposed to F. nucleatum in the presence and absence of PFBE containing 1.0 µg/mL of piceatannol at 60 min. Untreated cells served as control. Representative images from one experiment are shown. Indicator arrows show NF-κB accumulation within the nucleus of some cells.

Article Snippet: Subsequently, cells were blocked with a blocking buffer (nonfat dry milk; Bio-Rad Laboratories) at RT for 1 h. After washing, cells were incubated with rabbit anti-nuclear factor-kappa B (NF-κB) p65 primary antibody (1:400; Cell Signaling Technology, Danvers, MA, USA) at RT for 90 min and with CY3-conjugated goat anti-rabbit IgG secondary antibody (1:2000; Abcam, Cambridge, MA, USA) at RT for 45 min. NF-κB nuclear translocation was analyzed with the ZOE Fluorescent Cell Imager (Bio-Rad Laboratories).

Techniques: Immunofluorescence, Translocation Assay, Control