Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

doi: 10.1007/s00262-005-0698-x

Figure Lengend Snippet: IL-12 mRNA expression in melanoma xenograft isolated from mice treated with Virulizin. CD-1 nude mice bearing human melanoma C8161 tumors were treated with 0.2 ml of PBS or Virulizin for 4 weeks, and then tumors were collected 1 h after the last injection. RNA was extracted from tumor tissue by TRIZol reagent. The levels of IL-12α, IL-12β and β-actin mRNA were determined by quantitative real-time RT-PCR. IL-12 mRNA expression was normalized to β-actin level. Each bar represents average fold change calculated from five to seven mice. * p<0.05 compared to PBS control

Article Snippet: Anti-mouse biotinylated IL-12β antibody was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Techniques: Expressing, Isolation, Injection, Quantitative RT-PCR, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

doi: 10.1007/s00262-005-0698-x

Figure Lengend Snippet: Analysis of Intracellular IL-12 level in peritoneal macrophages by flow cytometry

Article Snippet: Anti-mouse biotinylated IL-12β antibody was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Techniques: Flow Cytometry, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

doi: 10.1007/s00262-005-0698-x

Figure Lengend Snippet: Increased level of IL-12β in sera of mice treated with Virulizin. CD-1 nude mice were treated with 0.2 ml of PBS or Virulizin for 3 days, and serum was collected 1 h after the last injection. IL-12 β in serum was determined by quantitative ELISA as described in Materials and Methods section. Each bar shows average levels calculated from ten mice. * p<0.05 compared to PBS control

Article Snippet: Anti-mouse biotinylated IL-12β antibody was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

doi: 10.1007/s00262-005-0698-x

Figure Lengend Snippet: Increased IL-12 mRNA expression in peritoneal macrophages from mice treated with Virulizin. CD-1 nude mice were treated intramuscularly with 0.2 ml of PBS or Virulizin daily for 5 days. Thioglycollate medium (1.5 ml, 3% w/v) was injected i.p. 2 days prior to macrophages collection. RNA was extracted from the cells by TRIZol reagent and IL-12α, IL-12β as well as β-actin genes were quantified by real-time RT-PCR. IL-12 mRNA expression was normalized to β-actin level. Each bar represents average fold change in IL-12 levels calculated from five to seven mice. * p<0.05 compared to PBS control

Article Snippet: Anti-mouse biotinylated IL-12β antibody was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Techniques: Expressing, Injection, Quantitative RT-PCR, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

doi: 10.1007/s00262-005-0698-x

Figure Lengend Snippet: Analysis of IL-12 intracellular level in peritoneal macrophages by flow cytometry. CD-1 nude mice were treated intramuscularly with 0.2 ml of PBS or Virulizin daily for 5 days. Thioglycollate medium (1.5 ml, 3% w/v) was injected i.p. 2 days prior to macrophages collection. The cells were stained with F4/80 antibody conjugated to FITC, biotinylated anti-IL-12 antibody and phycoerythrin (PE)-Cy5.5-conjugated streptavidin. Samples were examined by flow cytometry. a Histogram of flow cytometric analysis of the gated F4/80+ cells; b Percentage of IL-12α and β positive macrophages. The data are presented as the average of double positive cells (F4/80+ /IL-12+) (mean ± SE) calculated from three independent experiments in Table 1

Article Snippet: Anti-mouse biotinylated IL-12β antibody was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Techniques: Flow Cytometry, Injection, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

doi: 10.1007/s00262-005-0698-x

Figure Lengend Snippet: Neutralization of IL-12 in Virulizin-treated mice compromised NK cell cytotoxicity. CD-1 nude mice were treated with Virulizin or PBS daily for 3 days with or without one-time co-administration of 0.25 mg anti-IL-12 or control IgG per mouse. NK cells were isolated from the spleens one hour after the last injection, and co-cultured with YAC-1 target tumor cells to assess NK cell cytotoxicity using LDH release assay as shown in Fig. 2. The effector to target cell ratio used was 40:1. Each bar represents percent cytotoxicity (mean ± SE) calculated from three independent experiments

Article Snippet: Anti-mouse biotinylated IL-12β antibody was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Techniques: Neutralization, Control, Isolation, Injection, Cell Culture, Lactate Dehydrogenase Assay

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: Cardiac IL-12β expression was significantly increased after TAC. (A) Western blots and quantified data of LV IL-12β expression in WT sham and WT TAC mice. (B) Representative LV IL-12β staining images and quantified data of IL-12β + area. (C) Representative images of IL-12β and CD45 co-immunostaining of WT mice LV tissue under sham and TAC conditions. The green stain in panel B is due to cardiac autofluorescence recorded at FITC channel. ∗p < 0.05; n = 5 per group.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Expressing, Western Blot, Staining, Immunostaining

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO significantly attenuated TAC-induced cardiac dysfunction, an increase in LV weight, LA weight, lung weight, and RV weight in male and female mice. (A) Representative M-mode echocardiographic images of WT and IL-12β KO male mice: pre-TAC, 2 weeks, 4 weeks, and 6 weeks after TAC, and Quantified data of echocardiographic measurements of LVEF, LVFS, LVESD, and LVEDD of both sexes. (B) Survival curves of WT TAC and IL-12β KO TAC mice of both sexes (log-rank test). (C–F) The ratio of LV weight, left atrial (LA) weight, lung weight, RV weight to tibial length (TL) of the indicated groups. (G) Representative LV WGA staining images and quantified data of cardiomyocyte cross-sectional areas. ∗p < 0.05; # p < 0.05 compared to WT sham; † p < 0.05 compared to WT TAC; $ p < 0.05 compared to IL-12β KO sham; n = 7 to 22 per group for panels A-F and n = 4-5 per group for panel G; LVEF, LV ejection fraction; LVFS, LV fractional shortening; LVESD, LV end-systolic diameter; LVEDD, LV end-diastolic diameter.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Staining

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO attenuated TAC-induced alterations in the gene expression related to fibrosis and inflammation (A) Principal component analysis of WT and IL-12β KO mice, under sham or TAC conditions. (B) Venn diagram showing differentially expressed genes (DEGs) of WT and IL-12β KO mice under sham or TAC conditions, as well as the shared and uniquely changed genes. (C) DEGs cluster heatmap in WT mice compared to IL-12β KO mice, under sham or TAC conditions. (D) Volcano plots showing upregulated and downregulated genes among the groups. (E) Gene ontology (GO) biological processes enrichment bubble chart in WT TAC mice compared to IL-12β KO TAC. (F) GO cellular components enrichment histogram shows the enriched cellular components between KO and WT mice after TAC. n = 2 per group for sham and n = 3 per group for TAC.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Gene Expression

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO suppressed LV genetic pathways associated with extracellular matrix remodeling, inflammation, and LV fibrosis in mice after TAC. (A) The top 20 upregulated and downregulated pathways between KO and WT mice after TAC, identified by GSEA with ReactomeGSA. (B) Representative GSEA plots of collagen formation and extracellular matrix organization. (C, D) Representative images and quantified data of LV interstitial and perivascular fibrosis performed by Sirius Red/Fast Green staining in male and female mice. # p < 0.05 compared to WT sham; † p < 0.05 compared to WT TAC; $ p < 0.05 compared to IL-12β KO sham; n = 3 per group for panels A-B and n = 4-5 per group for panel D.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Staining

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO significantly attenuated TAC-induced LV inflammation in mice. (A) The top 20 upregulated and downregulated pathways between KO and WT mice after TAC were identified by GSEA with KEGG pathway analysis. (B) Heatmap shows the top LV immune and/or inflammation-related genes in KO and WT mice after TAC. (C, D) Representative LV CD45 + immuno-staining images and quantified data of LV CD45 + of the indicated groups. (E) Percentage distribution of major immune cell subsets within LV CD45 + cells of the indicated groups determined by flow cytometry. The green stain in panel C is due to cardiac autofluorescence recorded at FITC channel. # p < 0.05 compared to WT sham; † p < 0.05 compared to WT TAC; $ p < 0.05 compared to IL-12β KO sham; n = 2-3 per group for panels A-B and n = 4-5 per group for panels D-E.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Immunostaining, Flow Cytometry, Staining

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO attenuated TAC-induced pulmonary inflammation, fibrosis, and vessel remodeling in male and female mice. (A, B) Representative images and quantified data of infiltrated CD45 + leukocytes in the lung performed by immuno-histological staining. (C, D) Representative images and quantified data of lung fibrosis performed by Sirius Red/Fast Green staining. (E, F) Representative images and quantified data of lung vessel remodeling performed by immuno-histological staining. # p < 0.05 compared to WT sham; † p < 0.05 compared to WT TAC; $ p < 0.05 compared to IL-12β KO sham; n = 4-5 per group.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Staining

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO significantly attenuated TAC-induced early-phase LV dysfunction, cardiomyocyte hypertrophy, and inflammation. (A) Representative M-mode echocardiographic images and quantified data of echocardiographic measurements of LVEF, LVFS, LVESD, and LVEDD of the indicated groups. (B) The ratios of cardiac and pulmonary tissues to tibial length (TL) of the indicated groups. (C, D) Representative images and quantified data of LV cardiomyocyte size and LV CD45 + cells of the indicated groups. (E) Percentage of major immune cell subsets within LV CD45 + cells determined by flow cytometry. (F) Representative flow cytometry plots and quantified data of CD44 + CD3 + , CD44 + CD4 + , and CD44 + CD8 + T cells within the corresponding T cells. The green stain in panel D is due to cardiac autofluorescence recorded at FITC channel. ∗p < 0.05; n = 5-10 per group.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Flow Cytometry, Staining

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO significantly attenuated "stem cell-like" central memory T cell activation and their IFNγ production in the drainage lymph nodes. (A, B) Percentages of major immune cell subsets within CD45 + cells in the drainage lymph nodes determined by flow cytometry. (C) Percentages of IFNγ + CD8 + and TNFα + CD8 + within CD8 + T cells. (D, E) Percentages of CXCR3 + CD4 + and CXCR3 + CD8 + T cells within CD4 + and CD8 + T cells, respectively. (F) Percentage of effective memory (CD44 + CD62L − ), naïve (CD44 − CD62L + ), and central memory (CD44 + CD62L + ) T cells of CD8 + T cells. ∗p < 0.05; T EM , Effective Memory T Cells; T naïve , Naïve T Cells; T CM , Central Memory T Cells; n = 4-5 per group.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Activation Assay, Flow Cytometry

Journal: Redox Biology

Article Title: Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

doi: 10.1016/j.redox.2026.104082

Figure Lengend Snippet: IL-12β KO and anti-IL-12β antibody treatment significantly attenuated TAC-induced LV oxidative stress. (A) Representative dihydroethidium (DHE) staining images and quantified data of DHE intensity of the indicated groups. (B) Representative western blots and quantification of LV 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) of the indicated groups. (C-E) Representative DHE, 3-NT, and 4-HNE staining images, and quantified data of relative DHE, 3-NT, and 4-HNE intensity of the indicated groups. ∗p < 0.05; n = 5 to 6 per group.

Article Snippet: Anti-IL-12β antibody treatment: Pharmacological inhibition of IL-12β was achieved by anti-IL-12β antibody treatment (BioXCell, BE0051) in WT female mice.

Techniques: Staining, Western Blot

Journal: Foods

Article Title: Changes in Histological Structure, Interleukin 12, Smooth Muscle Actin and Nitric Oxide Synthase 1. and 3. Expression in the Liver of Running and Non-Running Wistar Rats Supplemented with Bee Pollen or Whey Protein

doi: 10.3390/foods11081131

Figure Lengend Snippet: ( a ) Con-Sed, ( b ) Con-Run, ( c ) WP-Sed, ( d ) BP-Sed, ( e ) WP-Run and ( f ) BP-Run; immuno-stainings against α-SMA; OD analysis of α-SMA showed increased expression in WP-Run group compared to the Con-Run group.

Article Snippet: The following antibodies were used: anti-α-Smooth Muscle Actin (α-SMA, Elabscience, Houston, TX, USA, E-AB-33323, dilution 1:200), anti-interleukin 12β (IL-12β, Biorbyt, orb184406, dilution 1:100), anti-nitric oxide synthase 1 (NOS1/nNOS, Elabscience, Houston, TX, USA, dilution 1:100), and anti-nitric oxide synthase 3 (NOS3/eNOS, Elabscience, Houston, TX, USA, dilution 1:100) and anti-transforming growth factor β (TGF–β, St John’s Laboratory, dilution 1:200).

Techniques: Expressing

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 1. IL-12α and EBI3 mutually promote their secretion and form IL-35. (A) IL- 35 (alphaFold2 docked model) shares its α subunit IL-12α with IL-12 [Protein Data Bank (PDB): 3HMX], and its β subunit EBI3 with IL-27 (PDB: 7u7n). To assess mutu- ally induced secretion of IL-12α and EBI3, constant DNA amounts of IL-12α were cotransfected with increasing DNA amounts of EBI3 (B) or vice versa (C). Both IL- 12α and EBI3 show a reduced mobility in the medium, indicating that both sub- units are retained in the endoplasmic reticulum in isolation and traverse the Golgi during secretion, as indicated by modification of their glycans. (D) Co-IP of FLAG- tagged IL-12α coexpressed with EBI3 in the cell medium verifies assembly for these two proteins. Quantification of the IL-12α IP efficiency and the fraction of EBI3 that is found in complex with IL-12α is shown (n = 3 ± SD). (E) Co-IP of secreted HA- tagged EBI3 coexpressed with IL-12αFLAG verifies assembly for these two proteins in the medium. Quantification of the EBI3HA IP efficiency and the fraction of IL- 12αFLAG that is found in complex with EBI3HA is shown (n = 5 ± SD). Constructs were expressed in human embryonic kidney (HEK) 293T cells. One representative immunoblot is shown in each case.

Article Snippet: Binding of the primary antibody was carried out overnight at 4°C with anti–IL12α (1:500 in MTBST; Abcam, ab133751), anti-IL-12β (1:500 in MTBST; Abcam, ab133752), EBI3 antisera (1:20 in PBS; provided by O. Devergne), anti-IL-27 (1:200 in MTBST; R&D Systems, AF2526), anti-HA tag (1:1000 in MTBST; BioLegend, 902301), anti-Hsc70 (1:1000 in MTBST; Santa Cruz Biotechnology, sc1059), or anti-His tag (1:1000 in MTBST; Proteintech, HRP66005).

Techniques: Isolation, Modification, Co-Immunoprecipitation Assay, Construct, Western Blot

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 2. IL-35 subunits can be secreted as nonheterodimers in contrast to IL-12 and IL-27. (A) EBI3 induces the secretion of IL-12α even when it is retained in the ER (via a C-terminal KDEL sequence). EBI3-induced secretion is observed for wild-type IL- 12α and a variant lacking the cysteine that forms an interchain disulfide bond in IL-12 (C96S). (B) The same as in (A), only that IL-12α was furnished with a KDEL ER-retention sequence and secretion of EBI3 was monitored. EBI3 secretion was slightly increased by coexpression with both IL-12α and the C96S variant. Quantifications of EBI3 secretion are shown below the blot. (C) A similar analysis for IL-12 reveals that IL-12βC199S, with an ER retention sequence, does not induce secretion of free IL- 12α but instead co-retains it in the cell. The same is observed for the combination of IL-12αC96S with IL-12βC199S, with both pro- teins lacking the cysteines that form the interchain disulfide bond in IL-12. (D) Coexpression of wild-type EBI3 and IL-27α leads to the secretion of IL-27. When IL-27α was ER-retained (IL- 27αKDEL), EBI3 secretion was reduced, in contrast to (B). (E) Free IL-12α can be detected in medium samples after pulldown of EBI3. IL-12α and EBI3 are cotransfected, and cell supernatants underwent two consecutive HA-IPs to isolate EBI3HA-containing complexes. IL-12α is also coimmunoprecipitated as can be seen in the IL-12α blot after HA-IP I and II, indicating pulldown of IL- 35. The final IL-12α–IP reveals remaining IL-12α in the medium that is not interacting with EBI3. Band intensities show a sig- nificantly higher amount of free IL-12α when coexpressed with EBI3 (or EBI3KDEL) compared to transfection in isolation without EBI3.

Article Snippet: Binding of the primary antibody was carried out overnight at 4°C with anti–IL12α (1:500 in MTBST; Abcam, ab133751), anti-IL-12β (1:500 in MTBST; Abcam, ab133752), EBI3 antisera (1:20 in PBS; provided by O. Devergne), anti-IL-27 (1:200 in MTBST; R&D Systems, AF2526), anti-HA tag (1:1000 in MTBST; BioLegend, 902301), anti-Hsc70 (1:1000 in MTBST; Santa Cruz Biotechnology, sc1059), or anti-His tag (1:1000 in MTBST; Proteintech, HRP66005).

Techniques: Sequencing, Variant Assay, Transfection, Isolation

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 3. Recombinant human IL-12αC96S and EBI3 are stable and well-structured proteins. (A) Analysis of purified IL-12αC96S and EBI3 by reducing and nonreducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Faster migration on nonreducing SDS-PAGE indicates the presence of disulfide bonds. Positions of intramolecular disulfide bonds are indicated in each subunit structure. (B) Reconstituted IL-12 and IL-27 are able to induce receptor heterodimerization. COS-7 cells were cotransfected with the indicated receptor chains equipped with the NanoBRET reporter system. Cells were stimulated with purified IL-12αC96S, which was previously incubated with recombinant human IL-12βC199S, or EBI3, previously incubated with murine IL-27α or the isolated subunits as indicated (10 nM final concentrations). Graphs represent the normalized NanoBRET signal (n = 3 ± SD). Statistical significance was calculated by one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test; ****P < 0.0001 compared with the corresponding phosphate-buffered saline (PBS) control. (C) NK-92 or BL-2 cells were stimulated with preincubated IL-12αC96S + IL- 12βC199S or EBI3 + mIL-27α or the heterodimeric cytokines (10 ng/ml final concentrations). Downstream signaling was detected by STAT-phosphorylation via immuno- blot. (D) Far-UV CD spectra for IL-12αC96S and EBI3. (E) IL-12αC96S unfolds cooperatively with an apparent melting temperature of 47 ± 0.2°C and EBI3 with an apparent melting temperature of 50 ± 0.2°C (green line, experimental data; black line, Boltzmann sigmoidal nonlinear curve fit; transitions were not reversible).

Article Snippet: Binding of the primary antibody was carried out overnight at 4°C with anti–IL12α (1:500 in MTBST; Abcam, ab133751), anti-IL-12β (1:500 in MTBST; Abcam, ab133752), EBI3 antisera (1:20 in PBS; provided by O. Devergne), anti-IL-27 (1:200 in MTBST; R&D Systems, AF2526), anti-HA tag (1:1000 in MTBST; BioLegend, 902301), anti-Hsc70 (1:1000 in MTBST; Santa Cruz Biotechnology, sc1059), or anti-His tag (1:1000 in MTBST; Proteintech, HRP66005).

Techniques: Recombinant, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Migration, Incubation, Isolation, Saline, Control, Phospho-proteomics, Circular Dichroism

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 4. IL-12aC96S and EBI3 act as immunosuppressors. (A) Concentrations of secreted IL-1β, IL-6, IL-8, and TNF-α [enzyme-linked immunosorbent assay (ELISA)] in supernatants from LPS stimulated human PBMCs after stimulation with IL-12aC96S or EBI3 (n = 10 donors). (B) Scheme of the experimental workflow to assess IL-12aC96S

Article Snippet: Binding of the primary antibody was carried out overnight at 4°C with anti–IL12α (1:500 in MTBST; Abcam, ab133751), anti-IL-12β (1:500 in MTBST; Abcam, ab133752), EBI3 antisera (1:20 in PBS; provided by O. Devergne), anti-IL-27 (1:200 in MTBST; R&D Systems, AF2526), anti-HA tag (1:1000 in MTBST; BioLegend, 902301), anti-Hsc70 (1:1000 in MTBST; Santa Cruz Biotechnology, sc1059), or anti-His tag (1:1000 in MTBST; Proteintech, HRP66005).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 5. IL-12αC96S and EBI3 show different sig- naling characteristics. (A) The NanoBRET re- porter system indicates IL-12 receptor heterodimerization after stimulation with 1 nM IL- 12, which can be blocked by a 30-min pretreat- ment with IL-12βC199S (1, 10, or 100 nM). (B) In the same assay, a 30-min pretreatment with IL- 12αC96S (1, 10, or 100 nM) shows reduced IL-12– induced receptor heterodimerization at a 10-fold excess of IL-12αC96S. (C) A preincubation with in- creasing concentration of EBI3 did not have any effect on IL-27 activity. Graphs represent the nor- malized NanoBRET signal (n = 3 ± SD). (D) STAT4 phosphorylation in NK-92 cells after treatment with IL-12 (1 ng/ml) is blocked by a 30-min pre- treatment with IL-12βC199S (10 and 100 ng/ml) or IL-12αC96S (10 ng/ml). Representative immuno- blots from one of three independent experiments are shown. (E) BL-2 cells were used to monitor IL- 27 (10 ng/ml)–mediated STAT1 phosphorylation, which is not blocked by pretreatment with EBI3 (10, 100, or 1000 ng/ml) for 30 min (n = 3 ± SD). (F) NK-92 or (G) BL-2 cells were pretreated with IL- 12αC96S (10 ng/ml) or EBI3 (10 ng/ml) overnight (o/n) before addition of IL-12 or IL-27, respec- tively, to investigate receptor internalization. (n = 3 ± SD, *P < 0.05 and ****P < 0.0001) (H) STAT1 phosphorylation in human PBMCs treated with EBI3 and IL-12αC96S compared to the cytokines IL- 10, IL-27, and IL-35 and their effect after HDM stimulation. Representative immunoblots from one of three independent experiments are shown.

Article Snippet: Binding of the primary antibody was carried out overnight at 4°C with anti–IL12α (1:500 in MTBST; Abcam, ab133751), anti-IL-12β (1:500 in MTBST; Abcam, ab133752), EBI3 antisera (1:20 in PBS; provided by O. Devergne), anti-IL-27 (1:200 in MTBST; R&D Systems, AF2526), anti-HA tag (1:1000 in MTBST; BioLegend, 902301), anti-Hsc70 (1:1000 in MTBST; Santa Cruz Biotechnology, sc1059), or anti-His tag (1:1000 in MTBST; Proteintech, HRP66005).

Techniques: Concentration Assay, Activity Assay, Phospho-proteomics, Western Blot

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 6. IL-12aC96S and EBI3 potentiate regulatory T cell development and suppress SEA-induced IL-4 production from human PBMCs. (A) Percentage of CD25hiFoxp3+ Treg cells [fluorescence-activated cell sorting (FACS)] in human PBMC cultures treated with IL-12aC96S (20 ng/ml) and EBI3 (20 ng/ml) or the com- bination of both IL-12aC96S and EBI3 (n = 8). Data are presented as means + SEM. Statistical significance was determined by Friedman test. *P < 0.05 and **P < 0.01. (B) Concentrations of IL-4 (ELISA) in culture supernatants from SEA-stimulated human PBMCs alone or in combination with IL-12C96S or EBI3 (n = 8 donors). The dotted line indicates mean secretion of IL-4 from PBS-treated PBMCs. (C) Concentrations of CCL17 (ELISA) in culture supernatants from IL-4–stimulated human PBMCs alone or in combination with IL-12C96S or EBI3 (n = 3 donors). (B and C) Data are presented as individual values. Donor-dependent effect is shown by the connecting line. Statistical significance was determined by Wilcoxon test. *P < 0.05 and **P < 0.01.

Article Snippet: Binding of the primary antibody was carried out overnight at 4°C with anti–IL12α (1:500 in MTBST; Abcam, ab133751), anti-IL-12β (1:500 in MTBST; Abcam, ab133752), EBI3 antisera (1:20 in PBS; provided by O. Devergne), anti-IL-27 (1:200 in MTBST; R&D Systems, AF2526), anti-HA tag (1:1000 in MTBST; BioLegend, 902301), anti-Hsc70 (1:1000 in MTBST; Santa Cruz Biotechnology, sc1059), or anti-His tag (1:1000 in MTBST; Proteintech, HRP66005).

Techniques: Fluorescence, FACS, Enzyme-linked Immunosorbent Assay