anti-mouse atf2  (Cell Signaling Technology Inc)

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

Journal: PLoS ONE

Article Title: Tumor Necrosis Factor-alpha utilizes MAPK/NFκB pathways to induce cholesterol-25 hydroxylase for amplifying pro-inflammatory response via 25-hydroxycholesterol-integrin-FAK pathway

doi: 10.1371/journal.pone.0257576

Figure Lengend Snippet: (A) A schematic diagram of the mouse C25H promoter (light yellow) and mouse C25H gene (orange) is shown. ATF2 binding region is shown in green along with the ATF2 binding sequence in the C25H promoter. The arrows with numbers represent the position of the forward and reverse primers used for the ChIP-qPCR assay. (B) Chromatin was isolated from MEFs treated with TNF (10ng/ml) for 4h. ChIP-qPCR assay was performed by using ATF2 antibody, control IgG and primers to amplify the genomic region flanking the ATF2 binding site in the C25H promoter. Location of the primers is shown in Fig 7a. ChIP-qPCR data is shown as Mean ± SEM from three independent experiments (n = 3). *p ≤ 0.05 using one-way ANOVA multiple comparison test.

Article Snippet: ChIP was performed with 2μg of mouse anti-ATF2 antibodies (Santa Cruz Biotechnology) or control mouse IgG (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Binding Assay, Sequencing, ChIP-qPCR, Isolation, Control, Comparison

Journal: Biochemical Journal

Article Title: Merkel cell polyomavirus small tumour antigen activates the p38 MAPK pathway to enhance cellular motility

doi: 10.1042/BCJ20200399

Figure Lengend Snippet: ( A ) HEK 293 cells were transfected with vectors that express MCPyV ST or empty vector for 48 h. Following immunoblotting, antibodies were used to detect phosphorylated or total ERK1/2 and p38. ST specific antibodies (2T2) were used to detect MCPyV ST and antibodies specific for GAPDH used as a loading control. ( B ) Lysates were immunoblotted to detect phosphorylated and total MK2, MSK1 and ATF2, with 2T2 and GAPDH used to confirm MCPyV ST expression and equal loading, respectively. Blots are representative of n = 3.

Article Snippet: Primary antibodies targeting P-ERK1/2, ERK1/2 total, P-p38, p38 total, P-MK2, MK2 total, P-MSK1, MSK1 total, P-ATF2, ATF2 total, P-MKK4, MKK4 total and P-MKK3/6 were purchased from Cell Signalling Technologies and diluted 1 : 1000 in tris buffered saline (pH 7.6), containing 1% tween 20 (TBS-T) and 5% BSA, with the exception of P-ERK1/2 which was diluted 1 : 2000.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing

Journal: Biochemical Journal

Article Title: Merkel cell polyomavirus small tumour antigen activates the p38 MAPK pathway to enhance cellular motility

doi: 10.1042/BCJ20200399

Figure Lengend Snippet: MCPyV ST interacts with PP4C to dysregulate its role in regulating the phosphorylation of either MKK4 or an upstream kinase. Following MCPyV ST binding of PP4C, MKK4 activation, in turn, phosphorylates p38, which in turn phosphorylates substrates including MK2, ATF2 and MSK1. The activation of downstream targets of p38 subsequently enhance migratory phenotypes associated with MCPyV ST.

Article Snippet: Primary antibodies targeting P-ERK1/2, ERK1/2 total, P-p38, p38 total, P-MK2, MK2 total, P-MSK1, MSK1 total, P-ATF2, ATF2 total, P-MKK4, MKK4 total and P-MKK3/6 were purchased from Cell Signalling Technologies and diluted 1 : 1000 in tris buffered saline (pH 7.6), containing 1% tween 20 (TBS-T) and 5% BSA, with the exception of P-ERK1/2 which was diluted 1 : 2000.

Techniques: Phospho-proteomics, Binding Assay, Activation Assay

Journal: Technology in Cancer Research & Treatment

Article Title: PPM1D Knockdown Suppresses Cell Proliferation, Promotes Cell Apoptosis, and Activates p38 MAPK/p53 Signaling Pathway in Acute Myeloid Leukemia

doi: 10.1177/1533033820942312

Figure Lengend Snippet: Antibodies Applied in Western Blot.

Article Snippet: ATF2 mouse mAb (sc-242) , Santa Cruz (USA) , 1:500.

Techniques: Western Blot

Journal: Technology in Cancer Research & Treatment

Article Title: PPM1D Knockdown Suppresses Cell Proliferation, Promotes Cell Apoptosis, and Activates p38 MAPK/p53 Signaling Pathway in Acute Myeloid Leukemia

doi: 10.1177/1533033820942312

Figure Lengend Snippet: PPM1D silencing activated p38 MAPK/p53 pathway in AML cells. The relative expression of p-p38, p-p53, and the target gene of p38 (p-ATF2), as well as target gene of p53 (Bax) in AML-193 cells (A and B) and KG-1 cells (C and D). AML indicates acute myeloid leukemia; PPM1D, protein phosphatase, Mg 2+ /Mn 2+ dependent 1D.

Article Snippet: ATF2 mouse mAb (sc-242) , Santa Cruz (USA) , 1:500.

Techniques: Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: PPM1D Knockdown Suppresses Cell Proliferation, Promotes Cell Apoptosis, and Activates p38 MAPK/p53 Signaling Pathway in Acute Myeloid Leukemia

doi: 10.1177/1533033820942312

Figure Lengend Snippet: Antibodies Applied in Western Blot.

Article Snippet: p-ATF2 mouse mAb (sc-8398) , Santa Cruz (USA) , 1:1000.

Techniques: Western Blot

Journal: Technology in Cancer Research & Treatment

Article Title: PPM1D Knockdown Suppresses Cell Proliferation, Promotes Cell Apoptosis, and Activates p38 MAPK/p53 Signaling Pathway in Acute Myeloid Leukemia

doi: 10.1177/1533033820942312

Figure Lengend Snippet: PPM1D silencing activated p38 MAPK/p53 pathway in AML cells. The relative expression of p-p38, p-p53, and the target gene of p38 (p-ATF2), as well as target gene of p53 (Bax) in AML-193 cells (A and B) and KG-1 cells (C and D). AML indicates acute myeloid leukemia; PPM1D, protein phosphatase, Mg 2+ /Mn 2+ dependent 1D.

Article Snippet: p-ATF2 mouse mAb (sc-8398) , Santa Cruz (USA) , 1:1000.

Techniques: Expressing