anti nf κb2 p100 p52  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: Histone decrotonylation plays a distinct role in HIV latency

doi: 10.1126/sciadv.aec0149

Figure Lengend Snippet: ( A ) Primary CD4 + T cell model of latency was activated with anti-CD3/CD28 Dynabeads. The percentage of GFP + cells was measured by flow cytometry (top). The levels of the ncNF-κB components p100 and p52, as well as ENL and CDYL were measured by Western blot (bottom) ( n = 3). The relative intensity was shown below with results normalized to dimethyl sulfoxide (DMSO) control. MW, molecular weight. ( B ) Primary CD4 + T cells from HIV-negative donors were infected with HIV-1 (SF162) and treated with or without 40 mM NaCr ( n = 3). HIV transcription levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting the HIV LTR, with results normalized to day 1 (left). The percentage of p24 + cells was measured by HIV-Flow assay (right). ( C ) TZM-bl luciferase reporter cells were transfected with empty vector (EV), Tat, wild-type (WT) HDAC3, or mutant HDAC3 (HDAC3-VRPP or HDAC3-Y298H), or Tat in combination with WT or mutant HDAC3 for 2 days ( n = 3). ( D ) A similar luciferase assay was performed with WT p300 and mutant p300-I1395G. Tat-induced HIV transcription was measured using a luciferase assay. Results are expressed as relative light units (RLU) and normalized to the EV control ( n = 4). The P values were determined using two-way analysis of variance (ANOVA) with multiple comparisons [(B), left], two-tailed unpaired Student’s t test [(B), right], or one-way ANOVA with multiple comparisons (C). Error bars represent SD; * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: Protein expression was assessed using the following antibodies: anti–NF-κB2 p100/p52 (Cell Signaling Technology), anti-ENL (Cell Signaling Technology), anti-CDYL (Gene Tex), anti–β-actin (Cell Signaling Technology), anti–α-tubulin (Cell Signaling Technology), anti–acetyl–α-tubulin (Cell Signaling Technology), anti-HDAC1 (Cell Signaling Technology), anti-HDAC2 (Cell Signaling Technology), anti-HDAC3 (Cell Signaling Technology), and anti-HDAC8 (Cell Signaling Technology).

Techniques: Flow Cytometry, Western Blot, Control, Molecular Weight, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Two Tailed Test

Journal: Cellular Oncology

Article Title: B3GNT3 facilitates NFKB2 processing and non-canonical NF-κB activation to drive lung adenocarcinoma progression

doi: 10.1007/s13402-026-01192-8

Figure Lengend Snippet: B3GNT3 interacts with NFKB2 and regulates cell cycle and EMT gene expression. ( A , B ) STRING interaction network ( A ) and functional enrichment analysis ( B ) of B3GNT3-interacting proteins identified by LC-MS/MS, highlighting a prominent association with the non-canonical NF-κB signaling pathway. ( C , D ) GSEA analysis showing significant enrichment of NF-κB 1( C ) and NF-κB2 ( D ) signaling-related gene signatures in PC9 cells overexpressing B3GNT3. ( E ) Co-immunoprecipitation showing interaction between Flag-tagged B3GNT3 and endogenous p100 (NFKB2) in PC9 cells. ( F ) Reciprocal co-immunoprecipitation demonstrating association between HA-tagged p100 and B3GNT3. ( G ) Glycosyltransferase activity assay of wild-type B3GNT3 and the catalytic-dead mutant B3GNT3 (D297A) using the UMP/CMP-Glo Glycosyltransferase Assay, confirming loss of enzymatic activity in the D297A mutant. ( H ) Co-immunoprecipitation analysis showing that the catalytic-dead mutant B3GNT3 (D297A) retains the ability to interact with p100 (NFKB2), with comparable binding to wild-type B3GNT3 in PC9 cells. ( I ) Western blot analysis of p100 phosphorylation and p52 generation in PC9 cells overexpressing B3GNT3 or B3GNT3 (D297A). ( J ) Nuclear-cytoplasmic fractionation analysis showing increased nuclear accumulation of p52 following B3GNT3 or B3GNT3 (D297A) overexpression. Wcl, whole cell lysate. Nuc, nucleus. Cyto, cytoplasm. ( K ) Quantification of nuclear and cytoplasmic p52 levels shown in ( J ). Band intensities were first normalized to Wcl input and subsequently calculated as the ratio of nuclear to cytoplasmic fractions

Article Snippet: Antibodies against pRB (s807/811, CST, 8516), Rb (CST, 9309) and p100 (s866/870, CST, 4810), p100 (CST, 4882) and H3 (CST, 9715) were purchased from Cell Signaling Tech.

Techniques: Gene Expression, Functional Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Activity Assay, Mutagenesis, Binding Assay, Western Blot, Phospho-proteomics, Fractionation, Over Expression

Journal: bioRxiv

Article Title: A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway

doi: 10.64898/2026.03.12.711255

Figure Lengend Snippet: A) Top-scoring transcription factors predicted to regulate the MYR1-dependent upregulated genes in HFF cells relative to uninfected controls were identified by analysis of previously published data using Enrichr. B-C) Representative images (top) and quantitative analysis (bottom) for RelB (B) and p52 (C) nuclear accumulation in HFFs infected with RH (WT), Δ myr1 or Δ myr1 ::MYR1 complement parasites. Twenty-four hours post-infection, cells were fixed and labeled with DAPI (blue), anti-GAP45 (red), and either anti-RelB or anti-p52 (green). Scale bars = 10 µm. Plots display the nuclear-to-cytoplasmic signal ratios for at least 300 infected cells (red arrows) per condition; uninfected cells are indicated by white arrows. Data from three independent experiments were combined for analysis. The horizontal dashed lines indicate the mean. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001, ns = not significant.

Article Snippet: Primary antibodies used were: rabbit anti-phospho-NF-kappaB2 p100 (Ser866/870) (1:1,000; Cell Signaling Technology, #4810), rabbit anti-NF-kappaB2 p100/p52 (1:1,000; Cell Signaling Technology, #4882), rabbit anti-NIK (1:1,000; Cell Signaling Technology, #4994), rabbit anti-TRAF3 (1:1,000; Cell Signaling Technology, #4729), and mouse anti-β-tubulin (Developmental Studies Hybridoma Bank, AB_528499), which detects both parasite and host β-tubulin and was used as a loading control.

Techniques: Infection, Labeling, Comparison

Journal: bioRxiv

Article Title: A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway

doi: 10.64898/2026.03.12.711255

Figure Lengend Snippet: A-B) Representative images (top) and quantitative analysis (bottom) for RelB (A) and p52 (B) nuclear accumulation in MEFs infected with RH (WT), Δ myr1 or Δ myr1 ::MYR1 complement parasites. Twenty-four hours post-infection, cells were fixed and labeled with DAPI (blue), anti-GAP45 (red), and either anti-RelB or anti-p52 (green). Scale bars = 10 µm. Plots display the nuclear-to-cytoplasmic signal ratios for at least 300 infected cells (red arrows) per condition; uninfected cells are indicated by white arrows. Data from three independent experiments were combined for analysis. The horizontal dashed lines indicate the mean. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001, ns = not significant.

Article Snippet: Primary antibodies used were: rabbit anti-phospho-NF-kappaB2 p100 (Ser866/870) (1:1,000; Cell Signaling Technology, #4810), rabbit anti-NF-kappaB2 p100/p52 (1:1,000; Cell Signaling Technology, #4882), rabbit anti-NIK (1:1,000; Cell Signaling Technology, #4994), rabbit anti-TRAF3 (1:1,000; Cell Signaling Technology, #4729), and mouse anti-β-tubulin (Developmental Studies Hybridoma Bank, AB_528499), which detects both parasite and host β-tubulin and was used as a loading control.

Techniques: Infection, Labeling, Comparison

Journal: bioRxiv

Article Title: A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway

doi: 10.64898/2026.03.12.711255

Figure Lengend Snippet: T. gondii activates the non-canonical NF-κB pathway through MYR1-dependent TRAF3 depletion and NIK stabilization. (A) Representative images (top) and quantitative analysis (bottom) for RelB nuclear accumulation in HFFs infected with RH (WT) parasites over a 24-h time course. At 6, 12, 18, and 24 h post-infection, cells were fixed and labeled with DAPI (blue), anti-GAP45 (red), and anti-RelB (green). Scale bars = 10 µm. Plots display the nuclear-to-cytoplasmic intensity ratios for at least 300 infected cells (red arrows) per condition; uninfected cells are indicated by white arrows. Data from three independent experiments were combined for analysis. The horizontal dashed lines indicate the mean. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparison test; ***P < 0.001, ****P < 0.0001, ns = not significant. (B) Immunoblot analysis (top) and corresponding quantification (bottom) of TRAF3, NIK, p100 phosphorylation, and p100-to-p52 processing in HFFs that were uninfected (UI) or infected with RH (WT), Δmyr1 or Δmyr1 ::MYR1 complement parasites. Lysates collected 24 h post-infection were resolved by SDS-PAGE and probed with specific primary antibodies; β-tubulin served as a loading control. Band intensities were measured using ImageJ and normalized to β-tubulin. Data are presented as mean ±SD from three biological replicates. Statistical significance for all panels was determined using a one-way ANOVA with Tukey’s multiple comparison test; **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant

Article Snippet: Primary antibodies used were: rabbit anti-phospho-NF-kappaB2 p100 (Ser866/870) (1:1,000; Cell Signaling Technology, #4810), rabbit anti-NF-kappaB2 p100/p52 (1:1,000; Cell Signaling Technology, #4882), rabbit anti-NIK (1:1,000; Cell Signaling Technology, #4994), rabbit anti-TRAF3 (1:1,000; Cell Signaling Technology, #4729), and mouse anti-β-tubulin (Developmental Studies Hybridoma Bank, AB_528499), which detects both parasite and host β-tubulin and was used as a loading control.

Techniques: Infection, Labeling, Comparison, Western Blot, Phospho-proteomics, SDS Page, Control