chip grade antibodies against nf κb  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM

doi: 10.1016/j.redox.2025.103996

Figure Lengend Snippet: Reg3g downregulates P2RX7 by promoting HSPG-NF-κB complex formation to maintain NF-κB in the cytoplasm. (A–C) The RAW264.7 cells were treated with Reg3g or PBS for 24 h in the presence of mEVs in these indicated experiments. (A) Representative NF-κB immunofluorescence (n = 3). (B) Western blot analysis of NF-κB in whole cell lysate (n = 3). (C) Western blot analysis of NF-κB in cell nucleus lysate (n = 3). (D) Predicted NF-κB binding site motif. (E) The putative NF-κB binding sites in P2rx7 promoter. The start site of transcription (TSS) is denoted +1 and the location of potential NF-κB binding site has been shown relative to TSS. (F–H) The RAW264.7 cells were treated with Reg3g in the presence of mEVs in these indicated experiments. (F) ChIP-qPCR analyzed the binding of NF-κB to P2rx7 promoter (n = 3 to 5). (G) Co-localization of HSPG and NF-κB. The line charts represent fluorescence intensity (MFI), which is presented the distance from α to ω in the images (n = 3). (H) The interaction of HSPG and NF-κB was determined by co-immunoprecipitation (n = 3). Data are presented as the means ± SEMs. For B and C, statistical significance was calculated using Student's unpaired two-tailed t -test. For F, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: DNA was sheared enzymatically and immunoprecipitated with the specific ChIP-grade antibodies against NF-κB (#8242, CST).

Techniques: Immunofluorescence, Western Blot, Binding Assay, ChIP-qPCR, Fluorescence, Immunoprecipitation, Two Tailed Test, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The NF-κB1/p50 Subunit Influences the Notch/IL-6-Driven Expansion of Myeloid-Derived Suppressor Cells in Murine T-Cell Acute Lymphoblastic Leukemia

doi: 10.3390/ijms25189882

Figure Lengend Snippet: The NF-κB1/p50 subunit represses the transcription of the IL-6 promoter in CD4 + CD8 + DP T-cells. ( A ) The binding of the NF-κB1/p50 or RELA/p65 subunit to the κB-consensus Site1 and Site3 of the murine IL-6 promoter , analyzed by ChIP-qPCR in cross-linked protein–DNA complexes of CD4 + CD8 + DP T thymocytes from wt mice, at 5–6 weeks of age. Fold enrichment of target region in p50-IP, p65-IP, or control IgG-IP is shown, and data are normalized to binding at the β-actin promoter (negative control). The values of IgG-IP controls were set to 1. ( B ) The binding of the RELA/p65 subunit to Site1 and Site3 of the murine IL-6 promoter, as in ( A ), analyzed by ChIP-qPCR in cross-linked protein–DNA complexes of CD4 + CD8 + DP T splenocytes from N3tg or N3tg / p50 −/− mice, compared to wt DP T thymocyte controls, at 5–6 weeks of age. Fold enrichment of target region in p65-IP versus control IgG-IP is shown, and data are normalized to binding at the β-actin promoter (negative control). The values in wt DP T thymocyte controls were set to 1. In ( A , B ), data represent the mean values ± SDs of three independent experiments ( n = 3 mice for each genotype), each in triplicate. ns = not significant, p > 0.05. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 represent significant differences between the indicated samples. ( C ) Working hypothesis on how the NF-κB1/p50 subunit can regulate IL-6 transcription and then MDSC differentiation in mice of different genotypes (see the text for an exhaustive explanation). In the left parts, x = no transcription, ++ = enhanced transcription and ++++ = very high transcription of the IL-6 promoter; vertical black arrows of different thickness indicate differences in the strength of p65 binding to the IL-6 promoter; blunt arrows of different thickness indicate differences in the inhibition of IL-6 transcription, that lacks in the panel 3 due to the absence of p50/p50 homodimers, as indicated by the red X over them. In the right parts, the triple red arrow indicates very high levels of IL-6 production by DP T cells; blunt arrows of different thickness indicate differences in the inhibition of CD8 + cells exerted by MDSCs, that lacks in the panel 1 due to the absence of IL-6 and MDSCs, as indicated by the red X over them. Created with BioRender.com.

Article Snippet: Then, chromatin immunoprecipitation was carried out with 5 μgr of antibodies against NF-κB1 p50 (E-10, sc-8414x, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ChIP-grade antibodies against NF-κB p65/RelA (17-10060, Millipore, Burlington, MA, USA), or an equivalent amount of normal-mouse IgG (12-371, Sigma-Aldrich) as a negative control.

Techniques: Binding Assay, ChIP-qPCR, Control, Negative Control, Inhibition