antibody against p50  (Proteintech)

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: GluN2A–NMDAR mediated neuronal NFκB activation plays a key role in exacerbating ischemic brain injury under hyperhomocysteinemic conditions

doi: 10.1177/0271678X251399012

Figure Lengend Snippet: Inhibition of GluN2A–NMDAR attenuates neuronal NFκB activation in HHcy rats following ischemia and RPF. Non-HHcy (control) and HHcy rats were subjected to sham surgery or MCAO for 60 min followed by RPF for 6 h. A subset of HHcy rats was treated with GluN2A–NMDAR inhibitor NVP-AAM077 at the onset of MCAO. (a, b, d, e) Tissue extracts from ipsilateral cortex were processed for mRNA analysis of NFκB subunits (a, d) p50 and (b, e) p65 by quantitative RT-PCR. Transcripts were normalized against HPRT and β-actin housekeeping genes as reference (mean ± SE, n = 6–7). Significant difference between means was assessed by one-way ANOVA and presented as * p < 0.05, ** p < 0.001, and *** p < 0.0001. (c, f) Coronal brain sections were processed for immunohistochemical staining with antibodies against the phosphorylated NFκB subunit (phospho-p65, red) and NeuN (green), along with DAPI (blue). Arrows indicate localization of phospho-p65 in NeuN positive cells. HHcy: hyperhomocysteinemic; RPF: reperfusion.

Article Snippet: The cDNA was used to perform quantitative RT-PCR using TaqMan Universal PCR Master Mix (Catalog #4304437; Applied Biosystems) and the following gene-specific TaqMan probes (ThermoFisher Scientific): COX2 (PTGS2)—Rn01483828_m1; NFκB1 (p50)—Rn01399572_m1; RelA (p65)—Rn01502266_m1; CD68—Rn01495634_g1; TNFα—Rn01525859_g1; β-actin—Rn00667869_m1; and HPRT1—Rn01527840_m1.

Techniques: Inhibition, Activation Assay, Control, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Denosumab attenuates knee osteoarthritis progression by inhibiting synovial inflammation via the RANK/TRAF6/FSTL1 signalling

doi: 10.1038/s41467-025-66202-z

Figure Lengend Snippet: A KEGG pathway analysis of DEGs, based on scRNA-seq. KEGG pathway enrichment was identified using a two-sided hypergeometric test. P -values were adjusted for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) correction. Terms with an adjusted P -value (FDR) < 0.05 were considered statistically significant. B Western blot analysis of FSTL1, TRAF6, IκB, phosphorylated IκB, p65 and p50 proteins in FLSs. Cells were stimulated with RANKL or RANKL + denosumab for 24 h ( n = 3, biological replicates). C Western blot analysis of p65 and p50 proteins in the cytoplasm and nucleus of FLSs. Cells were stimulated with RANKL or RANKL + denosumab for 24 h. Cyto, cytoplasm; Nuc, nucleus ( n = 3, biological replicates). D Representative co-immunoprecipitation analyses. Protein lysates of FLSs were immunoprecipitated with anti-TRAF6 or non-specific (ns) IgG after stimulation with RANKL, or RANKL + denosumab. The pulldown was subjected to immunoblotting analysis against RANK of the coimmunoprecipitated protein and equal amounts of the total protein lysates (input). Representative images are from three independent experiments. E Western blot analysis of FSTL1, TRAF6, IκB, phosphorylated IκB, p65 and p50 proteins in the indicated FLSs ( n = 3, biological replicates). F Western blot analysis of p65 and p50 proteins in the cytoplasm and nucleus of indicated FLSs ( n = 3, biological replicates). G Schematic diagram of RANK/TRAF6/FSTL1-mediated NF-κB signalling regulated by RANKL. Created in BioRender. Hu, Y. (2025) https://BioRender.com/vva2i57 . H Representative immunofluorescence images of p65 (green) and p50 (red) in the indicated FLSs. Blue: DAPI showing total FLSs. Scale bar = 100 μm. Representative images are from three independent experiments. I Western blotting analysis of ADAMTS5, MMP-13 and TNF-α in the indicated FLSs ( n = 3, biological replicates). Source data and P values are provided in the Source Data file. Data are presented as median with interquartile range. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test for multiple comparisons ( B , C , E , F ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells cultured on slides were fixed with 4% PFA for 20 min at room temperature then incubated with a primary antibody against p50 (1:100, Proteintech, 14220-1-AP) or p65 (1:100, Proteintech, 10745-1-AP), CD86 (1:100, Proteintech, 13395-1-AP) or Arg1 (1:100, Affinity Biosciences, DF6657), overnight at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence

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: 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: The remaining chromatin was immunoprecipitated overnight at 4 °C with 5 μg primary antibody against Hif1α (GeneTex, GTX127309), NF-κB p50 (Proteintech, 14220-1-AP), Histone H3 (CST, #4620) or rabbit IgG (CST, #2729).

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