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: The following primary antibodies were used: rabbit anti-RelB (1:300; Proteintech, 25027-1-AP), mouse anti-NFκB p52 (1:100; Santa Cruz Biotechnology, sc-7386), and mouse anti-GAP45 or rabbit anti-GAP45 (kindly provided by Dr D. Etheridge, University of Georgia, USA).

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: The following primary antibodies were used: rabbit anti-RelB (1:300; Proteintech, 25027-1-AP), mouse anti-NFκB p52 (1:100; Santa Cruz Biotechnology, sc-7386), and mouse anti-GAP45 or rabbit anti-GAP45 (kindly provided by Dr D. Etheridge, University of Georgia, USA).

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: The following primary antibodies were used: rabbit anti-RelB (1:300; Proteintech, 25027-1-AP), mouse anti-NFκB p52 (1:100; Santa Cruz Biotechnology, sc-7386), and mouse anti-GAP45 or rabbit anti-GAP45 (kindly provided by Dr D. Etheridge, University of Georgia, USA).

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

Journal: Frontiers in Immunology

Article Title: IFNγ-mediated suppression of alternative NF-κB in tumor-resident myeloid cells promotes selective recruitment of cytotoxic but not regulatory T cells

doi: 10.3389/fimmu.2025.1681777

Figure Lengend Snippet: The induction of CCL22, but not CTL attractants, uniquely depends on both canonical and alternative NF-κB signaling. Macrophages were stimulated with recombinant human TNFα and/or IFNγ for 24 hours in the absence or presence of selective NF-κB inhibitors (given 2 hours prior to stimulation). 24-hour culture supernatants were analyzed by ELISA for CTL-attractants (A) or Treg attractants (B) . All data shown are mean +/- SEM of triplicate cultures from the same donor representing one of three independent experiments with different donors. ns, not significant; **p < 0.005; ***p < 0.0005; ****p < 0.0001.

Article Snippet: The following antibodies were purchased for these studies: NF-κB p65/RelA (Santa Cruz Biotechnology Cat# sc-8008, RRID: AB_628017), NF-κB p52/p100 (Santa Cruz Biotechnology Cat# sc-7386, RRID: AB_2267131), CCL5 (Bd Biosciences Cat# 564754, RRID: AB_2738932), CXCL10 (BD Biosciences Cat# 555049, RRID: AB_395670), CD8 (BD Biosciences Cat# 563823, RRID: AB_2687487), CD33 (BD Biosciences Cat# 551378, RRID: AB_398502), CD4 (BD Biosciences Cat# 555347, RRID: AB_395752), Granzyme B (BD Biosciences Cat# 560211, RRID: AB_1645488), FoxP3 (BioLegend Cat# 320124, RRID: AB_2565972), and CD326 (BioLegend Cat# 324208, RRID: AB_756082).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: IFNγ-mediated suppression of alternative NF-κB in tumor-resident myeloid cells promotes selective recruitment of cytotoxic but not regulatory T cells

doi: 10.3389/fimmu.2025.1681777

Figure Lengend Snippet: IFNγ selectively suppresses alternative NF-κB signaling. After 1 hour ( (A) to visualize canonical NF-κB translocation) or 24 hours ( (B) to visualize alternative NF-κB translocation) of stimulation in the indicated conditions, macrophages were fixed, permeabilized, and stained for NF-κB proteins p65 (canonical) and p52 (alternative). Nuclear localization of NF-κB proteins was quantified by imaging cytometry using median Similarity Score analysis (histograms) and normalized using the Fisher’s Discriminant ratio (bar graphs). Bar graphs present the mean +/- SEM of six independent experiments with different donors. Representative images for key conditions are displayed on the right side of the histograms (40X magnification). *p < 0.05.

Article Snippet: The following antibodies were purchased for these studies: NF-κB p65/RelA (Santa Cruz Biotechnology Cat# sc-8008, RRID: AB_628017), NF-κB p52/p100 (Santa Cruz Biotechnology Cat# sc-7386, RRID: AB_2267131), CCL5 (Bd Biosciences Cat# 564754, RRID: AB_2738932), CXCL10 (BD Biosciences Cat# 555049, RRID: AB_395670), CD8 (BD Biosciences Cat# 563823, RRID: AB_2687487), CD33 (BD Biosciences Cat# 551378, RRID: AB_398502), CD4 (BD Biosciences Cat# 555347, RRID: AB_395752), Granzyme B (BD Biosciences Cat# 560211, RRID: AB_1645488), FoxP3 (BioLegend Cat# 320124, RRID: AB_2565972), and CD326 (BioLegend Cat# 324208, RRID: AB_756082).

Techniques: Translocation Assay, Staining, Imaging, Cytometry