mouse anti phospho atf2  (Santa Cruz Biotechnology)

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: Anti-mouse ATF2 , Cell Signaling Technology , Cat# 9226 S.

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: Reagents and tools table

Article Snippet: Anti-mouse ATF2 , Cell Signaling Technology , Cat# 9226 S.

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

Journal: bioRxiv

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

doi: 10.1101/2023.08.04.551982

Figure Lengend Snippet: ( A ) mRNA expression of p35 subunit of IL-35 in human visceral fat isolated from lean (N=12) and obese patients (N=52). ( C ) C57BL6 mice were treated with recombinant IL-35 i.v. (300ng per mouse) and BAT temperature was measured 4h later (mean ± SEM; PBS n = 7; IL-35 treated mice n = 9). ( 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 48h and UCP1 and FGF21 levels were analysed by immunoblot. Loading control for UCP1 was run on different gel and not presented. (n=4 for each condition, technical replicates). ( 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 minutes and ATF2 phosphorylation was analysed by immunoblot. ( F ) Differentiated adipocytes were stimulated with IL-35 (100 ng/ml) for 48h in the presence or absence of SB203580 inhibitor (10 uM). 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 IL-35 (100 ng/ml) for 48h in the presence or absence of SB203580 inhibitor (10 uM). The expression of principal adipogenic markers ( Pparg, Adipoq, Leptin, Perlinipin ) level was measured by qRT-PCR and relativized to b-actin . Data are mean ± SEM, *p < 0.05, ** p < 0.01, ***p<0.001. Analysis by t test (A, C, E, F), 2-way ANOVA (B) or 1-way ANOVA (G).

Article Snippet: Primary antibodies used in the study: anti-mouse MKK3 (Cat# 9238, Cell Signaling Technology), anti-mouse MKK6 (Cat# ADI-KAP-MA014-E, Enzo Life Sciences), anti-mouse UCP1 (Cat# AB10983, Abcam), anti-mouse FGF21 (Cat# RD281108100, BioVendor), anti-mouse p-ATF2 T69/71 (Cat# 9225S, Cell Signaling Technology), anti-mouse ATF2 (Cat# 9226S, Cell Signaling Technology), anti-mouse p-s6 S240/244 (Cat# 5364S, Cell Signaling Technology), anti-mouse p-p38 T180/Y182 (Cat# 9211S, Cell Signaling Technology), anti-mouse b-actin (Cat# sc-47778, Santa Cruz Technology), anti-mouse vinculin (Cat# V9131, Sigma) and secondary antibodies used in the study: goat anti-mouse (Cat# 31430, ThermoFisher) and goat anti-rabbit (Cat# 31460, ThermoFisher).

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

Journal: bioRxiv

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

doi: 10.1101/2023.08.04.551982

Figure Lengend Snippet: T cell expression of stress kinases MKK3 and MKK6 contributes to obesity and inflammation in AT through a compromised metabolic profile characterized by reduced browning and thermogenesis, as well as worsened liver steatosis. When T cells lack p38 activation, it enhances the expansion of adipose tissue Tregs and increases Treg IL35 expression via the mTOR pathway. IL35 induces phosphorylation of ATF-2, leading to the upregulation of UCP1 and FGF21 levels in adipocytes, which in turn promotes browning. Additionally, IL35 limits the infiltration and inflammation of CD8 + T cells in adipose tissue, thereby providing protection against obesity.

Article Snippet: Primary antibodies used in the study: anti-mouse MKK3 (Cat# 9238, Cell Signaling Technology), anti-mouse MKK6 (Cat# ADI-KAP-MA014-E, Enzo Life Sciences), anti-mouse UCP1 (Cat# AB10983, Abcam), anti-mouse FGF21 (Cat# RD281108100, BioVendor), anti-mouse p-ATF2 T69/71 (Cat# 9225S, Cell Signaling Technology), anti-mouse ATF2 (Cat# 9226S, Cell Signaling Technology), anti-mouse p-s6 S240/244 (Cat# 5364S, Cell Signaling Technology), anti-mouse p-p38 T180/Y182 (Cat# 9211S, Cell Signaling Technology), anti-mouse b-actin (Cat# sc-47778, Santa Cruz Technology), anti-mouse vinculin (Cat# V9131, Sigma) and secondary antibodies used in the study: goat anti-mouse (Cat# 31430, ThermoFisher) and goat anti-rabbit (Cat# 31460, ThermoFisher).

Techniques: Expressing, Activation Assay, Phospho-proteomics