il 35 protein  (MedChemExpress)

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-23p19 and IL-27/IL-35Ebi3 can form a heterodimer, termed IL-39. (A) Detection and characterization of human IL-39 by immunoprecipitation (IP) and Western blot (WB) analyses under reducing conditions. (B) Schematic of the cDNA constructs used to genetically engineer mouse p19 and Ebi3 proteins. hPGK: human phosphoglycerate kinase 1 promoter. (C) Detection and characterization of mouse IL-39 recombinant proteins by IP and Western blot analyses under reducing conditions. (D) Coomassie Blue gel of the recombinant mouse IL-39 characterized on nonreducing polyacrylamide gels. Arrow indicates recombinant IL-39. (A, C, and D) Blots are representative of three independent experiments.

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Immunoprecipitation, Western Blot, Construct, Recombinant

Journal: EMBO Reports

Article Title: Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis

doi: 10.1038/s44319-024-00149-y

Figure Lengend Snippet: ( A ) mRNA expression of p35 subunit of IL-35 in human visceral fat isolated from lean and obese patients. ( B ) MKK3/6 CD4-KO and control CD4-Cre mice were exposed to cold for 4 h. Body and BAT temperature was measured every hour. ( C ) C57BL6 mice were treated with recombinant IL-35 i.v. (300 ng per mouse) and BAT temperature was measured 4 h later ( D ) Immortalized brown preadipocytes were differentiated in vitro. Once differentiated, cells were stimulated in the presence or absence of IL-35 (100 ng/ml) for 48 h and UCP1 and FGF21 levels were analyzed by immunoblot. Loading control for UCP1 was run on different gel and not presented. ( E ) Immortalized brown preadipocytes were differentiated in vitro. Once differentiated, cells were stimulated in the presence or absence of IL-35 (100 ng/ml) for 0–120 min and ATF2 phosphorylation was analyzed by immunoblot. ( F ) Differentiated adipocytes were stimulated with IL-35 (100 ng/ml) for 48 h in the presence or absence of SB203580 inhibitor (10 μM). The expression of Ucp1 level was measured by qRT-PCR and relativized to b-actin . ( G ) Primary white preadipocytes were isolated from C57BL6 mice and differentiated in vitro. Once differentiated, cells were stimulated with PBS or with IL-35 (100 ng/ml) for 48 h in the presence or absence of SB203580 inhibitor (10 μM). The expression of principal adipogenic markers ( Pparg , Adipoq , Leptin, Perlinipin ) level was measured by qRT-PCR and relativized to b-actin . Data Information: Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Exact p -values are shown. Analysis by t test ( A , C , D , F ), 2-way ANOVA ( B ), or 1-way ANOVA ( G ). Lean N = 12 biologically independent patients; Obese N = 52 biologically independent patients ( A ). n = 5–9 biologically independent mice ( B , C ) or n = 2–6 biologically independent wells ( D – G ) for each group, represented as single dots in the graphs. .

Article Snippet: Differentiated adipocytes were stimulated in the presence or absence of 100 ng/ml IL-35 (010-001-B66, Rockland) for 0–2 h or for 48 h and then the cells were lysed for western blot.

Techniques: Expressing, Isolation, Control, Recombinant, In Vitro, Western Blot, Phospho-proteomics, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis

doi: 10.1038/s44319-024-00149-y

Figure Lengend Snippet: ( A – C ) MKK3/6 CD4-KO and CD4-Cre mice were fed a high-fat diet (HFD) for 8 weeks. ( A , B ) qRT-PCR analysis of mRNA expression in ( A ) eWAT and ( B ) SVF isolated from control or MKK3/6 CD4-KO mice. mRNA expression was normalized to b-actin mRNA. ( C ) FACS quantification and representative dot plots of IL-35 + Treg cells in lymph nodes. ( D ) In vitro Treg cell induction (iTreg). Naive CD4 + T cells were isolated from the spleens of CD4-Cre and MKK3/6 CD4-KO mice stimulated for 96 h with plate-bound anti-CD3, soluble anti-CD28 + IL-2 + TGFβ. qRT-PCR analysis of p35 and Ebi3 mRNA in iTregs derived from control or MKK3/6 CD4-KO mice. mRNA expression was normalized to b-actin mRNA. ( E ) Induction of iTregs from CD4 + T cells isolated from healthy human donor buffy coats and stimulated with plate-bound anti-CD3, soluble hIL-2 + hTGFβ for 6 days in the presence or absence of the p38 pan inhibitor BIRB796. qRT-PCR analysis of P35 and EBI3 mRNA in iTregs. mRNA expression was normalized to GAPDH mRNA. ( F ) FACS analysis of IL-35 MFI in in vitro induced iTreg cells from CD4-Cre and TSC1 CD4-KO mice. ( G ) Western blot analysis of p-s6 protein S240/244 and p-p38 Thr180/Tyr182 in iTreg cells from CD4-Cre and MKK3/6 CD4-KO mice. Loading control for p-p38 was run on different gel and not presented. ( H ) FACS analysis of IL-35 MFI in in vitro induced iTreg cells from MKK3/6 CD4-KO mice in the presence or absence of rapamycin for 4 h. Data Information: Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant. Exact p-values are shown. Analysis by t test. n = 4–10 biologically independent mice ( A – C ) or n = 4–9 biologically independent wells ( D – H ) for each group, represented as single dots in the graphs. .

Article Snippet: IL-35 EBI3 Mouse Recombinant Protein , Rockland , Cat# 010-001-B66.

Techniques: Quantitative RT-PCR, Expressing, Isolation, Control, In Vitro, Derivative Assay, Western Blot

Journal: EMBO Reports

Article Title: Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis

doi: 10.1038/s44319-024-00149-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: IL-35 EBI3 Mouse Recombinant Protein , Rockland , Cat# 010-001-B66.

Techniques: Recombinant, Sequencing, SYBR Green Assay, Marker, Purification, Western Blot, Software, Staining, Cell Isolation, Reverse Transcription, Transgenic Assay, Membrane, Electrophoresis, Microscopy