phosphorylated-p65 (ser536) antibody  (Cell Signaling Technology Inc)

Journal: World Journal of Clinical Oncology

Article Title: Natural compound rosmarinic acid displays anti-tumor activity in colorectal cancer cells by suppressing nuclear factor-kappa B signaling

doi: 10.5306/wjco.v16.i5.105341

Figure Lengend Snippet: Rosmarinic acid inhibits nuclear factor-kappa B signaling in colorectal cancer cells. A: Molecular modeling of rosmarinic acid (RA)/inhibitory kappa B kinase beta complex. 3D presentation of the molecular docking complex pose (I), 2D presentation (II), and 3D presentation (III) of the interactions between RA and the key residues of inhibitory kappa B kinase beta; B and C: HT29 (B) and SW480 (C) cells transfected with p-nuclear factor-kappa B-Luc along with Renilla luciferase were incubated with indicated RA overnight, followed by luciferase assay; D: HT29 and SW480 cells were incubated with increasing concentrations of RA for 24 hours, followed by Western blot analysis using antibodies against p-p65 and nuclear factor-kappa B P65. GAPDH was used as a loading control; E: Starved HT29 cells were treated with indicated RA for 12 hours, and then cells were incubated with 200 ng/mL lipopolysaccharide for 30 minutes, followed by Western blot analysis using antibodies against p-p65 and GAPDH; F and G: HT29 cells were incubated with increasing concentrations of RA for 24 hours, followed by quantitative real-time polymerase chain reaction to evaluate the mRNA levels of cyclin D1 (F) and MYC (G). GAPDH was used as an internal control. n = 3. Data are shown as the mean ± SD. a P < 0.001 vs group ‘0’, b P < 0.0001 vs group ‘0’. RA: Rosmarinic acid; NF-κB: Nuclear factor-kappa B; LPS: Lipopolysaccharide.

Article Snippet: Anti-phosphorylated nuclear factor-kappa B (NF-κB) P65 (p-p65, Ser536) antibody, anti-NF-κB P65 antibody, anti-Bcl-2 antibody, anti-caspase-3 antibody, and anti-phosphorylated protein kinase B (p-AKT, Ser473) antibody were purchased from Cell Signaling Technology, Danvers, MA, United States.

Techniques: Transfection, Luciferase, Incubation, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: HDAC6 inhibition by ITF3756 modulates PD-L1 expression and monocyte phenotype: insights for a promising immune checkpoint blockade co-treatment therapy

doi: 10.3389/fimmu.2025.1546939

Figure Lengend Snippet: Signaling pathways involved in PD-L1 regulation by ITF3756 in TNF-α stimulated monocytes. Purified human monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml). (A) Analysis of the modulation by TNF-α (left panel) and by the combination of TNF-α and ITF3756 (right panel) of specific pathways involved in PD-L1 regulation. RNAseq data obtained as described before were used for this analysis. Significant differentially expressed genes are represented as circles, while non-significant genes are shown as triangles. (B) Transcription factor activity inference upon treatment with TNF-α (left panel) and with the combination of TNF-α and ITF3756 (right panel). (C) Cytofluorimetric analysis of p65 phosphorylation on Ser536 (p-p65). Monocytes were pre-treated for 2h with ITF3756 and then stimulated or not with TNF-α for 15 minutes. RM one-way ANOVA followed by Dunnetts multiple comparison test was used for the statistical analysis. The graphs show the results obtained from 6 different donors. *p<0,05, **p<0,001, ***p<0,0005.

Article Snippet: After incubation cells were washed and permeabilized with BD PhosFlow Perm Buffer III (BD Bioscience) on ice for 30min, washed twice with PBS-0.1% FBS and incubated with anti-phosphorylated p65 Ser536 Alexafluor488 (Cell Signaling) antibody and human FcR blocking for 1h at RT.

Techniques: Protein-Protein interactions, Purification, Activity Assay, Phospho-proteomics, Comparison