p65 primary antibody 764s  (Cell Signaling Technology Inc)

Journal: Genes & Diseases

Article Title: Exogenous pyruvate is therapeutic against colitis by targeting cytosolic phospholipase A2

doi: 10.1016/j.gendis.2025.101571

Figure Lengend Snippet: Pyruvate inhibits TNFα/NF-κB signaling and the expression of downstream pro-inflammatory genes. (A) BMDMs isolated from WT mice were treated with TNFα in the absence (control) or presence of different metabolites for 24 h. Total RNA was extracted for quantitative reverse transcription PCR analysis for various inflammatory genes. The expression in the TNFα group, normalized against Gapdh, is regarded as 1. (B – D) RAW264.7 cells were incubated with TNFα in the absence or presence of various concentrations of metabolites shortlisted from (A), as indicated, for 24 h. The mRNA levels of (B) IL-6, (C) IL-1β, and (D) CCL2 were detected by quantitative real-time PCR ( n = 6). (E) NFκB-Luc mice induced with colitis were used to confirm the in vivo anti-inflammatory activity of 9 metabolites isolated from the cell-based preliminary screening. After treatment for 5 days with indicated metabolites, the luciferase reporter signal was detected by an in vivo imaging system. Representative bioluminescence images were acquired on the 7th day of the experimental time. All images were captured 10 min post-substrate administration with a 60-s exposure. (F) Quantification of panel E, representing light emitted ventrally from the mice post-luciferin injection. Statistical analysis was done to compare the treatment groups and control groups ( n = 6 mice for each group). Data are mean ± standard error of the mean; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (G, H) mRNA expression of IL-1β and IL-6 with increasing concentrations of pyruvate (as indicated). (I, J) mRNA expression of IL-1β and IL-6 with low (2 mM) or high (4 mM) concentration of pyruvate. (K, L) The concentration of IL-1β (pg/mL) and IL-6 (pg/mL) in the supernatant was measured by ELISA. Statistical analysis was done to compare the treatment groups and vehicle groups ( n = 3 mice per group). Data are mean ± standard error of the mean; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (M) BMDMs were cultured with TNFα (10 ng/mL) in the absence or presence of pyruvate for 6 h. Confocal microscopy was performed to visualize the nuclear translocation of p65. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Scale bar, 100 μm. n = 3 mice per group. (N) Quantification of (M), illustrating the nuclear translocation in corresponding groups. TNFα, tumor necrosis factor-alpha; NF-κB, nuclear factor-kappa B; BMDMs, bone marrow-derived macrophages; WT, wild type; IL-6, interleukin-6; IL-1β, interleukin-1beta; CCL2, C–C motif chemokine ligand 2.

Article Snippet: After incubation, the cells were fixed with 4% paraformaldehyde solution at room temperature for 10 min, and then permeabilized in 0.1% Triton X-100 for 10 min, followed by blocked in 1% BSA at room temperature for 1 h. Cells were then incubated with p65 primary antibody (764s, cell Signaling) with dilution 1:50–1:100 at 4 °C overnight.

Techniques: Expressing, Isolation, Control, Reverse Transcription, Incubation, Real-time Polymerase Chain Reaction, In Vivo, Activity Assay, Luciferase, In Vivo Imaging, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Confocal Microscopy, Translocation Assay, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Pharmacological inhibition of CXCR4 increases the anti-tumor activity of conventional and targeted therapies in B-cell lymphoma models

doi: 10.1101/2025.07.10.664047

Figure Lengend Snippet: REC1 cells were exposed to DMSO (control), SPX5551 (4 µM), ibrutinib (4 nM), or a combination of the two for 6 hours. Representative images of two independent experiments and protein quantification of RELA (cytoplasmatic, A) or RELB (nuclear, B). DAPI is stained blue, and RELA/RELB is stained red. Barplots on the left show the median fluorescence intensity, * for p<0.05, ** for p<0.01 from a Mann-Whitney U-test.

Article Snippet: It was followed by the incubation of samples at 4°C with primary rabbit monoclonal anti-human NF-kB p65 antibody (D14E12) (1/100; Cell Signaling) overnight.

Techniques: Control, Staining, Fluorescence, MANN-WHITNEY