mab7624  (R&D Systems)

Journal: bioRxiv

Article Title: CK2 inhibition suppresses glial inflammation in the brain

doi: 10.1101/2025.08.05.668554

Figure Lengend Snippet: A. Knockdown efficiency of experiments shown in (mean expression shown with each dot representing an experiment). B. Western blotting showing expression of CK2Α1 and GAPDH in individual THP-1 NF-κB-Lucia clones, quantified as a relative ratio. Heterozygotes and homozygotes confirmed by Sanger sequencing. Full blots in . C. Western blotting showing CK2Α2 expression in individual WT or KO THP-1 NF-κB-Lucia clones after immunoprecipitation of CK2Α2. Full blots in . D. Quantification of NF-κB reporter luciferase activity in CK2Α1 heterozygous (het) or homozygous (KO) knockout THP-1 NF-κB Lucia cell clones stimulated with 20 ng/mL LPS and CHR dilution series (mean ± sem, n = 3). E. Expression of CK2α1-HA and CK2α2-HA in GPCs confirmed by Western blotting. Full blots in . F. CK2 kinase-dead mutants block IL1β-induced IL6 upregulation in HCA (3 independent experiments shown with Poisson error; relative expression normalized to each WT). G. Quantification showing that pIκB/IκBα levels are reduced with CK2 inhibitors (6-hour treatment, mean ± SD, n = 2, pooled independent experiments; related to ). H. Immunoblot and quantification (n = 2, mean) of NF-κB IP showing NF-κB S529 phosphorylation is reduced with CHR (2-hour treatment). Full blots in .

Article Snippet: Primary Antibodies used: Chicken anti-GFP (1:300; Aves Labs: GFP-1020), Goat polyclonal anti-hSox9 (1:250; R&D Biosystems: AF3075), Rabbit monoclonal anti-NF-κB p65/RelA(1:400; CTS: 8242), Mouse monoclonal anti-Phospho-RelA/NFkB p65 (S529) (1:50; R&D Systems: MAB7624), Rabbit polyclonal anti-CSNK2A2 (1:100; Proteintech: 10606-1-AP); Chicken polyclonal anti-NeuN (1:250; Millipore: ABN91).

Techniques: Knockdown, Expressing, Western Blot, Clone Assay, Sequencing, Immunoprecipitation, Luciferase, Activity Assay, Knock-Out, Blocking Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: CK2 inhibition suppresses glial inflammation in the brain

doi: 10.1101/2025.08.05.668554

Figure Lengend Snippet: A. CK2 levels increase with inflammation (time course by Western blot). Full blots in . B. Representative immunofluorescence images and quantification showing reduction of nuclear phospho-CK2α1 Y255/CK2α1 after 5 hours of CK2 inhibitor treatment (mean ± s.e.m., n = 4, one-way ANOVA with Dunnett’s post-hoc test). Scale bar = 100 µm. C. Representative immunoblots showing that pIκB/IκBα levels are reduced with CK2 inhibitors (6-hour treatment). Full blots in . D. Representative immunofluorescence images and quantification showing reduction of nuclear phospho-NF-κB S529/NF-κB after 5 hours of CK2 inhibitor treatment (mean ± s.e.m., n = 4, one-way ANOVA with Dunnett’s post-hoc test). Scale bar = 100 µm. E. Quantification of NF-κB reporter luciferase activity in a pool of phosphodeficient THP-1 NF-κB Lucia NF-κB-S529A-NeoR knockin cells or parental WT stimulated with various doses of LPS (mean of n = 3 biological replicates shown). F. X2K interaction network showing top enriched kinase modules, intermediate proteins (not labeled), and their downstream TF targets in IL1-β-stimulated astrocytes (gray nodes and edges unrelated to CK2, black nodes connected to CK2 via red edges). G. Immune signatures are significantly enriched in genes downregulated in inflamed astrocytes treated with API. H. CK2 inhibition reduces expression of “Astro-Inflammation” genes and increases expression of “Astro-Injury” genes. A-E: Representative of 3 independent experiments unless otherwise noted. ns = not significant; * P < 0.05; ** P < 0.01

Article Snippet: Primary Antibodies used: Chicken anti-GFP (1:300; Aves Labs: GFP-1020), Goat polyclonal anti-hSox9 (1:250; R&D Biosystems: AF3075), Rabbit monoclonal anti-NF-κB p65/RelA(1:400; CTS: 8242), Mouse monoclonal anti-Phospho-RelA/NFkB p65 (S529) (1:50; R&D Systems: MAB7624), Rabbit polyclonal anti-CSNK2A2 (1:100; Proteintech: 10606-1-AP); Chicken polyclonal anti-NeuN (1:250; Millipore: ABN91).

Techniques: Western Blot, Immunofluorescence, Luciferase, Activity Assay, Knock-In, Labeling, Inhibition, Expressing

Journal: Frontiers in Immunology

Article Title: BTLA agonist attenuates Th17-driven inflammation in a mouse model of steroid-resistant asthma

doi: 10.3389/fimmu.2025.1552394

Figure Lengend Snippet: HDM-driven airway inflammation in BXD75 mice are enriched for genes related to Th17 mediated inflammation. (A) Volcano plot of differentially expressed genes (DEGs) in C57BL/6 vs BXD75 mice challenged with HDM only. (B) Gene set enrichment analysis by Ingenuity Pathway Analysis (IPA) depicting enriched pathways in HDM-challenged BXD75 mice. (C) Heatmap representation of co-receptors in CD4 + T cells isolated from BXD75 mice vs C57BL/6 mice challenged with HDM. n = 3 mice per genotype. Mean fluorescence intensity (MFI) and representative flow cytometry histograms of (D) HVEM, (E) LIGHT, and (F) BTLA in CD4 + T cells from C57BL/6 and BXD75 mice challenged with HDM. (G) Schematic of downstream HVEM-LIGHT stimulatory signaling in T cells. MFI of (H) TRAF2, (I) p65, (J) p52, and (K) NIK in CD4 + T cells from HDM or PBS challenged C57BL/6 and BXD75 mice. Data in (D–F) are from one experiment that is representative of three independent experiments. Data in (H–K) are pooled from two experiments. Each experiment was performed at least three times. For all quantifications, data are presented as mean ± SEM and analyzed with a two-way ANOVA with Tukey’s multiple comparison test. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.0001.

Article Snippet: Staining for the NFkB pathway was done with the following antibodies: PE-anti-mouse TRAF2 (Clone H-10, Santa Cruz Biotechnology), PE anti-mouse NIK (Clone A-12, Santa Cruz Biotechnology), Alexa ® Fluor 647 anti-mouse NFκB p52/p100/NFKB2 Antibody (Clone C-5, Santa Cruz Biotechnology), PE anti-human/mouse p65/RelA (Clone 532301, R&D Systems).

Techniques: Isolation, Fluorescence, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: BTLA agonist attenuates Th17-driven inflammation in a mouse model of steroid-resistant asthma

doi: 10.3389/fimmu.2025.1552394

Figure Lengend Snippet: BTLA agonist treatment attenuates Th17 activation in vitro . (A) Naïve CD3 + CD4 + CD44 low T cells were isolated from C57BL/6 mice and cultured with bound anti-CD3, soluble CD28, rmIL-6, rmTGF- β1, rmIL-23, anti-IL-12, anti-IL-4 and anti-IFNγ for 5 days. On day 3, cells were treated with 20 μg/mL of BTLA agonist (Clone 6A6) or Isotype. (B) The number of CD4 + IL - 17A + T cells derived from C57BL/6 mice. (C) Levels of IL-17A in the cell culture supernatant. (D) Representative flow cytometry histogram and (E) quantification of SHP-1 expression MFI on Day 5. (F) Representative flow cytometry histogram and (G) quantification of p65 expression MFI on Day 5. (H) Representative flow cytometry histogram and (I) quantification of NIK expression MFI on Day 5. (J) Representative flow cytometry histogram and (K) quantification of p52 expression MFI on Day 5. Histograms are from one experiment that is representative of three independent experiments. Data in (B, C, E, I) are pooled from two experiments. Data in (G, K) are from one representative experiment. Each experiment was performed at least three times. For all quantifications, data are presented as mean ± SEM and analyzed with a two-tailed Student’s t test. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.0001.

Article Snippet: Staining for the NFkB pathway was done with the following antibodies: PE-anti-mouse TRAF2 (Clone H-10, Santa Cruz Biotechnology), PE anti-mouse NIK (Clone A-12, Santa Cruz Biotechnology), Alexa ® Fluor 647 anti-mouse NFκB p52/p100/NFKB2 Antibody (Clone C-5, Santa Cruz Biotechnology), PE anti-human/mouse p65/RelA (Clone 532301, R&D Systems).

Techniques: Activation Assay, In Vitro, Isolation, Cell Culture, Derivative Assay, Flow Cytometry, Expressing, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: SIRPα engagement regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism

doi: 10.1038/s41423-024-01208-z

Figure Lengend Snippet: SIRPα modulates ILC2 mitochondrial respiration via NF-κB pathways. A – P Cohorts of WT and SIRPα KO mice were intranasally challenged with rmIL-33 over 3 consecutive days. On day 4, lung ILC2s were isolated and cultured with rmIL-2 and rmIL-7 for 24 h. B Total RNA was extracted to perform a bulk transcriptomic analysis. Volcano plots represent differentially expressed genes. C Gene set enrichment analysis by Ingenuity Pathway Analysis (IPA) depicting critical pathways regulated in SIRPα-deficient ILC2s. D Dot plot representation of selected critical genes involved in ILC2 related genes, MAPK, and JAK/STAT pathways. Dot size is indicative of the total gene expression level. E Overview of downstream SIRPα signaling elements. F – I Cohorts of WT and SIRPα KO mice were challenged intranasally for 3 days with rmIL-33. On day 4, lung ILC2s were isolated and cultured with rmIL-2, rmIL-7 for 24 h. Representative histogram of protein expression of pSTAT3 ( G ), p38 ( H ), KLF2 ( I ), and p65 ( J ). Corresponding quantitation is presented as MFI; n = 4. Corresponding quantitation is presented for each protein as MFI; n = 4. K Dot plot representation of selected critical genes involved in OXPHOS and Mitochondrial respiratory pathways. Dot size is indicative of the total gene expression level. L – O Mitochondrial respiratory profile showing oxygen consumption rates (OCR) in response to sequential injections of Oligomycin (ATP synthase inhibitor), BAM15 (mitochondrial uncoupler), and Rotenone + antimycin A (complex I and II inhibitors). Key parameters of mitochondrial function, including basal respiration ( M ), spare respiratory capacity ( N ), and ATP production rate ( O ) are presented; n = 3. P Mitochondrial sizes were assessed using Mito Tracker green and are shown in plot graphs. Data are presented as mean +/− SEM and are representative of at least 2 experiments. Two-tailed student’s t-test was employed for statistical analysis; *< 0.05, **< 0.01, ***< 0.001, and ns= non-significant. Schematic images are sourced by an open-access license from Servier Medical Art

Article Snippet: Phosphorylation assessment involved FITC anti-human/mouse Phospho STAT3 (RUO, Biosciences), PE anti-human/mouse Rela (p65) (532301, R&D systems), APC anti-human/mouse Phospho p38 (4NIT4KK, Invitrogen), and Alexa FluorTM 647 anti-human/mouse KLF2 (bs-2772R, Bioss).

Techniques: Isolation, Cell Culture, Gene Expression, Expressing, Quantitation Assay, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: SIRPα engagement regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism

doi: 10.1038/s41423-024-01208-z

Figure Lengend Snippet: CD47 administration controls ILC2 function via activating SIRPα signaling. A )WT (CD45.1) and CD47 KO (CD45.2) mice were intranasally exposed to 0.5 μg rmIL-33 for three days, and lung ILC2s were isolated on day four. Two groups were formed: one consisting ILC2s from only CD47 KO mice (monoculture group), and another consisting of ILC2s from both CD47 KO (CD45.2) and WT (CD45.1) mice, which were co-cultured in a 1:1 ratio (co-culture group) and incubated for 24 h. The function of CD47 KO-derived ILC2 in each group was then evaluated by FACS. B , C Expression levels of GATA-3 ( B ) and Ki67 ( C ) in activated ILC2s are presented. Corresponding quantitation is presented as MFI; n = 4. D Frequency (%) of IL-5 + and IL-13 + ILC2s in both monoculture and co-culture groups; n = 4. E – I WT mice received intranasal doses of rmIL-33 over 3 consecutive days. Activated ILC2s were sorted and cultured with rmIL-2 (10 ng/ml) and rmIL-7 (10 ng/ml) in the presence of either vehicle or CD47-Fc (20 μg/mL) for 24 h. F , G GATA-3 ( F ) and Ki67 ( G ) expression levels in activated ILC2s are shown. Corresponding quantitation is presented as MFI; n = 4. H , I Levels of IL-5 ( H ) and IL-13 ( I ) production in the culture supernatant were measured by LEGENDPLEX and are shown in bar graphs; n = 4. J WT mice were treated with rmIL-33 by intranasal injection for three days in a row. Activated ILC2 cells were sorted and cultured with rmIL-2 (10 ng/ml) and rmIL-7 (10 ng/ml). The following day, vehicle or CD47-Fc (20 μg/mL) was added to the culture wells and analyzed by FACS after one hour. K Overview of downstream SIRPα signaling elements. L – O Representative histogram of protein expression of pSTAT3 ( L ), p38 ( M ), KLF2 (N ), and p65 ( O ). Corresponding quantitation is presented for each protein as MFI; n = 4. Data are presented as means ± SEM and are representative of at least 2 independent experiments. Two-tailed student’s t-test or one-way ANOVA followed by Tukey post-hoc tests were employed for statistical analysis; *< 0.05, **< 0.01, ***< 0.001, and ****< 0.0001. Schematic images are sourced by an open access license from Servier Medical Art

Article Snippet: Phosphorylation assessment involved FITC anti-human/mouse Phospho STAT3 (RUO, Biosciences), PE anti-human/mouse Rela (p65) (532301, R&D systems), APC anti-human/mouse Phospho p38 (4NIT4KK, Invitrogen), and Alexa FluorTM 647 anti-human/mouse KLF2 (bs-2772R, Bioss).

Techniques: Isolation, Cell Culture, Co-Culture Assay, Incubation, Derivative Assay, Expressing, Quantitation Assay, Injection, Two Tailed Test

Journal: Cancers

Article Title: Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells

doi: 10.3390/cancers12071904

Figure Lengend Snippet: HPV16 HPV E7 promotes nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation in oral cells. ( A ) pHAGE/NF-κB reporter vector map. ( B ) Luciferase activity normalized with GFP was evaluated in SCC143/E7 and SCC143/V cells transfected with the reporter vector pHAGE/NF-κB. ( C ) Luciferase activity normalized with GFP in SCC143/E7 cells co-transfected with pHAGE/NF-κB and siRNA for PIR or siRNA E7 knockdown and siRNA (SCR) as a control. ( D ) Protein array of NF-κB signaling pathway comparing the SCC143/V and SCC143/E7 cell extracts. Data were plotted in reference to the change that occurred in the presence of E7 compared to the empty vector control. ( E ) Western blot of nuclear and cytoplasmic protein fractions was performed to analyze the levels of Pirin, p65 S529 , p65, and C-Rel. β-actin or H3 were used as a load control in SCC143 cells transduced with empty (pLXSN) or E7 constructs. The graphs represent a densitometric analysis of three independent Western blots (WBs) for each protein normalized against β-actin. Data are presented as the mean ± SEM; average of three independent experiments, conducted in triplicate. * p < 0.05 (Mann–Whitney test).

Article Snippet: Membranes were incubated for 1 h at room temperature with blocking buffer (5% bovine serum albumin, Tris-buffered saline (TBS)–0.5% Tween 20, pH 7.6) and incubated overnight at 4 °C with primary antibody against Pirin (ab51360), pRb (ab24), EGFR (ab32562), EGFR Y1173 (ab32578), EGFR Y1068 (ab40815), p65 s536 (ab86299), β-actin (ab6276) (Abcam, Cambridge, UK), C-Rel (MAB2699), p65 s529 (MAB7624) (R&D System, Minneapolis, MN, USA), HPV16 E7 (SC6981), pAKT1-2-3 (SC514032), PHOSPHO AKT S473 (4060S), NRF2 (4060S) (Cell Signaling, Danver, MA, USA), ERK (SC514302), pERK (SC136521), AREG (SC5797), pMTORC (SC293133), MTORC (SC517464), Histone H3 (SC56616), GSK3 (SC7291), p65 (SC8008), (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1/1000 in Tris-buffered saline–Tween 20 (TBS–T20).

Techniques: Activation Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Knockdown, Control, Protein Array, Western Blot, Transduction, Construct, MANN-WHITNEY

Journal: Cancers

Article Title: Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells

doi: 10.3390/cancers12071904

Figure Lengend Snippet: HPV16 HPV E7 induces EGFR/PI3K/AKT1 signaling for PIR/NF-kB activation in oral cells. ( A ) Western blot was performed with protein extracts from SCC143/E7 cells previously exposed to Gefitinib (EGFR), U0126 (ERK) and LY294002 (PI3K) inhibitors for 24 h. The levels of total EGFR, pEGFR Y1173 , AKT1, pAKT1, ERK, pERK, Pirin and β-actin used as load control were analyzed. The graphs represent a densitometric analysis of three independent WBs for Pirin normalized against β-actin. ( B ) Time–response assay by exposure to Gefitinib for 1.5 to 24 h in SCC143/E7 cells. The levels of total EGFR, pEGFR Y1068 , Pirin and β-actin used as load control were analyzed. The graph represents a densitometric analysis of three independent experiments. ( C ) Time–response assay by exposure to 10 µM LY294002 for 3 to 24 h in SCC143/E7 cells. The levels of total AKT1, pAKT1, Pirin and β-actin used as load control were analyzed. The graph represents densitometric analysis of three independent experiments. ( D ) Western blot to evaluate AKT1, pAKT1, Pirin protein levels in organotypic raft cultures established from OKF6/TERT2 E7 oral cells treated with dimethyl sulfoxide (DMSO) or Gefitinib for 3 h (β-actin used as load control were analyzed). ( E ) Western blot to evaluate AKT1, pAKT1, Pirin protein levels in OKF6/TERT2 E7 oral organotypic raft culture cells treated with DMSO or 10 µM LY294002 for 12 or 24 h (β-actin used as load control were analyzed). ( F ) Western blot to evaluate c-Rel and p65 S529 protein levels in organotypic raft cultures established from OKF6/TERT2 E7 oral cells treated with DMSO or 10 µM LY294002 for 12 or 24 h (β-actin used as load control were analyzed) ( G ) Luciferase activity normalized against GFP was evaluated in SCC143/E7 cells transfected with the reporter vector pHAGE/NF-κB treated with DMSO or 10 µM LY294002 for 12 or 24 h. Data are presented as the mean ± SEM; average of three independent experiments, conducted in triplicate. * p < 0.05 and ** p < 0.01 (ANOVA test). LY294002 (LY).

Article Snippet: Membranes were incubated for 1 h at room temperature with blocking buffer (5% bovine serum albumin, Tris-buffered saline (TBS)–0.5% Tween 20, pH 7.6) and incubated overnight at 4 °C with primary antibody against Pirin (ab51360), pRb (ab24), EGFR (ab32562), EGFR Y1173 (ab32578), EGFR Y1068 (ab40815), p65 s536 (ab86299), β-actin (ab6276) (Abcam, Cambridge, UK), C-Rel (MAB2699), p65 s529 (MAB7624) (R&D System, Minneapolis, MN, USA), HPV16 E7 (SC6981), pAKT1-2-3 (SC514032), PHOSPHO AKT S473 (4060S), NRF2 (4060S) (Cell Signaling, Danver, MA, USA), ERK (SC514302), pERK (SC136521), AREG (SC5797), pMTORC (SC293133), MTORC (SC517464), Histone H3 (SC56616), GSK3 (SC7291), p65 (SC8008), (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1/1000 in Tris-buffered saline–Tween 20 (TBS–T20).

Techniques: Activation Assay, Western Blot, Control, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Cancers

Article Title: Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells

doi: 10.3390/cancers12071904

Figure Lengend Snippet: HPV16 E7 expression and PIR induces migration of oral cells. ( A ) A migration assay in SCC143/E7 and SCC143/V cells was carried out for 7 h using fibronectin pretreated transwells, scale bar 25 µm. ( B ) A migration assay performed on SCC143/E7 cells previously transfected with control siRNA (SCR), siRNA PIR or HPV16 siRNA E7 was carried out for 7 h using fibronectin pretreated transwells, scale bar 25 µm. ( C ) A migration assay performed on SCC143/E7 cells previously transfected with control siRNA (SCR) and siRNA I-II p65 was carried out for 7 h using fibronectin pretreated transwells, scale bar 25 µm. ( D ) Western blot against eGFP performed for V corresponding to the empty vector (pcDNA 3.1–eGFP) and PIR corresponding to the vector containing the PIR sequence linked to eGFP (pcDNA 3.1–eGFP–PIR) transfected in SCC143 oral cell. ( E ) A migration assay in SCC143 V (pcDNA 3.1–eGFP) and SCC143 PIR (pcDNA 3.1–eGFP–PIR) cells was carried out for 7 h using fibronectin pretreated transwells. Scale bar: 40 µm. ( F ) Analysis of E-cadherin and N-cadherin protein levels, which were normalized with the expression of β-actin. The graphs represent a densitometric analysis of three independent assays. Data are presented as the mean ± SEM; average of three independent experiments, conducted in triplicate. * p < 0.05 and ** p < 0.01 (Mann–Whitney test).

Article Snippet: Membranes were incubated for 1 h at room temperature with blocking buffer (5% bovine serum albumin, Tris-buffered saline (TBS)–0.5% Tween 20, pH 7.6) and incubated overnight at 4 °C with primary antibody against Pirin (ab51360), pRb (ab24), EGFR (ab32562), EGFR Y1173 (ab32578), EGFR Y1068 (ab40815), p65 s536 (ab86299), β-actin (ab6276) (Abcam, Cambridge, UK), C-Rel (MAB2699), p65 s529 (MAB7624) (R&D System, Minneapolis, MN, USA), HPV16 E7 (SC6981), pAKT1-2-3 (SC514032), PHOSPHO AKT S473 (4060S), NRF2 (4060S) (Cell Signaling, Danver, MA, USA), ERK (SC514302), pERK (SC136521), AREG (SC5797), pMTORC (SC293133), MTORC (SC517464), Histone H3 (SC56616), GSK3 (SC7291), p65 (SC8008), (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1/1000 in Tris-buffered saline–Tween 20 (TBS–T20).

Techniques: Expressing, Migration, Transfection, Control, Western Blot, Plasmid Preparation, Sequencing, MANN-WHITNEY