Anti-ATF2 antibody [E243] - ChIP Grade

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

Immunocytochemistry analysis of A549 (human lung carcinoma epithelial cell) labeling ATF2 with purified ab32160 at 1/100 dilution (10 µg/ml). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.10 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control : PBS instead of the primary antibody.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ATF2 (red) with ab32160 at a 1/2000 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

ab32160 at a dilution of 1/250 staining ATF2 in paraffin embedded breast carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ChIP

Unknown

ChIP - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

Chromatin was prepared from Jurkat (TPA and Ionomycin treated or not) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin 5μg of ab32160 (red) and 20μl of protein A/G sepharose beads slurry (10μl of sepharose A beads + 10μl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• IP

Unknown

Immunoprecipitation - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

ab32160 (purified) at 1/1000 dilution (2.284 © : g/ml) immunoprecipitating ATF2 in HeLa whole cell lysate.
Lane 3 (-) : HeLa(Human cervix adenocarcinoma epithelial cell) whole cell lysate 10© : gab32160 & HeLa whole cell lysateRabbit monoclonal IgG (ab172730) instead of ab32160 in HeLa whole cell lysate
For western blotting, Rabbit TureBlot : Anti-Rabbit IgG HRP was used as the secondary antibody at 1/1500 dilution.
Blocking and diluting buffer : 5% NFDM /TBST .

All lanes:

Immunoprecipitation - Anti-ATF2 antibody [E243] - ChIP Grade (ab32160)

Predicted band size: 55 kDa

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• WB

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Western blot - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

All lanes:

Western blot - Anti-ATF2 antibody [E243] - ChIP Grade (ab32160) at 1/10000 dilution

All lanes:

HeLa cell lysate

Predicted band size: 55 kDa

Observed band size: 70 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ K-562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab ab32160[E243]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ K-562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab ab32160[E243]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ K-562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab ab32160[E243]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

CiteAb

Western blot - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

Western Blotting using Anti-ATF2 antibody [E243] - ChIP Grade, ab32160. Publication image from Sun, B. et al., 2016, Nat Commun, 27853137. Legend direct from paper.

TGF-β increases CUGBP1 expression in HSCs via p38 MAPK.(a) Quantitative PCR analyses of CUGBP1 mRNA from the human hepatic stellate cell line LX-2 or human hepatocyte L02 cells treated with or without 5 ng ml−1 TGF-β for 6 h (mean±s.e.m.; n=3, **P<0.01 by Student's t-test). (b,c) Western blot analyses of CUGBP1 from LX-2 cells, primary mouse HSCs (b), and primary mouse hepatocytes (c) treated with or without 5 ng ml−1 TGF-β for 24 h. The data are representative of three independent experiments (mean±s.e.m.; n=3, **P<0.01 by Student's t-test). (d,e) LX-2 cells were treated with actinomycin D (1 µg ml−1) and with or without 5 ng ml−1 TGF-β for indicated time intervals (d). LX-2 cells were treated with or without SB431542 (10 µM), SB203580 (10 µM), SP600125 (10 µM), FR180204 (10 µM) or SIS3 (20 µM), following 5 ng ml−1 TGF-β treatment for 6 h (e). And then quantitative PCR was carried out to detect the remaining mRNA expression of CUGBP1. (mean±s.e.m.; n=3, *P<0.05, **P<0.01 by one-way analysis of variance followed by Dunnett's test). (f) Western blot analyses of LX-2 cells treated with or without SB431542, following 5 ng ml−1 TGF-β treatment for 24 h. (g) Gene2promotor analyses of promoter and transcription factors of human CUGBP1 gene. (h) Probe pull down assay was performed by mixing CUGBP1-CRE-Bio or mCUGBP1-CRE-Bio with total cell extracts from LX-2 cells treated as in f. Precipitates were prepared for Western blotting using SoftLink Soft Release avidin resin. The data in f and h are representative of two independent experiments.

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• WB

CiteAb

Western blot - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

Western Blotting using Anti-ATF2 antibody [E243] - ChIP Grade, ab32160. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

Pharmacological inactivation of mitochondrial copper(II) attenuates inflammation in vivo.a, Western blots of copper-signalling effectors in SPMs from mice treated with LPS. Macrophages of several mice were pooled (4–7 mice per condition). b, Western blots of copper-signalling effectors in SPMs from mice subjected to CLP. Macrophages of several mice were pooled (7–8 mice per condition). H3 is a sample processing control. c, Western blots of copper-signalling effectors in AMs from K18-hACE2 mice infected with SARS-CoV-2. Macrophages of several mice were pooled (10 mice per condition). H3 is a sample processing control. d, Average body temperature of mice treated as indicated (n = 6–9 mice per group). e, GO term analysis of downregulated genes in lung tissues of SARS-CoV-2 infected K18-hACE2 mice treated with LCC-12 (0.5 mg/kg). f, RNA-seq analysis of gene expression in lung tissues of SARS-CoV-2-infected K18-hACE2 mice treated with LCC-12 (0.5 mg/kg) (n = 8 mice per group). Inflammatory signature genes highlighted. Dashed lines, adjusted P value = 0.05. g, Illustration of copper-signalling. Cell plasticity involves upregulation of the cell surface marker CD44, which mediates endocytosis of metal-bound hyaluronates. In the presence of copper(II), NADH reacts with H2O2 to replenish NAD+ in mitochondria, an enzyme cofactor involved in the biosynthesis ofαKG and acetyl-CoA. These co-substrates of iron-dependent demethylases and acetyl-transferases are required for epigenetic and transcriptional programming of inflammation and the regulation of cell plasticity. Pharmacological inactivation of mitochondrial copper(II) blocks NAD(H) redox cycling, leading to distinct epigenetic states and transcriptional profiles. Targeting copper(II) interferes with cell plasticity in immune and cancer cells. For a – c gating strategy of SPMs and AMs see Methods and Supplementary Information. For d 2-way ANOVA. Mean values ± s.e.m. For e and f differential gene expression was assessed with the limma/voom framework. GO enrichment was assessed with the enrichGO method from clusterProfiler. P-values were corrected for multiple testing with the Benjamini-Hochberg procedure.Source data

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• WB

CiteAb

Western blot - Anti-ATF2 antibody [E243] - ChIP Grade (AB32160)

Western Blotting using Anti-ATF2 antibody [E243] - ChIP Grade, ab32160. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

Pharmacological inactivation of mitochondrial copper(II) attenuates inflammation in vivo.a, Western blots of copper-signalling effectors in SPMs from mice treated with LPS. Macrophages of several mice were pooled (4–7 mice per condition). b, Western blots of copper-signalling effectors in SPMs from mice subjected to CLP. Macrophages of several mice were pooled (7–8 mice per condition). H3 is a sample processing control. c, Western blots of copper-signalling effectors in AMs from K18-hACE2 mice infected with SARS-CoV-2. Macrophages of several mice were pooled (10 mice per condition). H3 is a sample processing control. d, Average body temperature of mice treated as indicated (n = 6–9 mice per group). e, GO term analysis of downregulated genes in lung tissues of SARS-CoV-2 infected K18-hACE2 mice treated with LCC-12 (0.5 mg/kg). f, RNA-seq analysis of gene expression in lung tissues of SARS-CoV-2-infected K18-hACE2 mice treated with LCC-12 (0.5 mg/kg) (n = 8 mice per group). Inflammatory signature genes highlighted. Dashed lines, adjusted P value = 0.05. g, Illustration of copper-signalling. Cell plasticity involves upregulation of the cell surface marker CD44, which mediates endocytosis of metal-bound hyaluronates. In the presence of copper(II), NADH reacts with H2O2 to replenish NAD+ in mitochondria, an enzyme cofactor involved in the biosynthesis ofαKG and acetyl-CoA. These co-substrates of iron-dependent demethylases and acetyl-transferases are required for epigenetic and transcriptional programming of inflammation and the regulation of cell plasticity. Pharmacological inactivation of mitochondrial copper(II) blocks NAD(H) redox cycling, leading to distinct epigenetic states and transcriptional profiles. Targeting copper(II) interferes with cell plasticity in immune and cancer cells. For a – c gating strategy of SPMs and AMs see Methods and Supplementary Information. For d 2-way ANOVA. Mean values ± s.e.m. For e and f differential gene expression was assessed with the limma/voom framework. GO enrichment was assessed with the enrichGO method from clusterProfiler. P-values were corrected for multiple testing with the Benjamini-Hochberg procedure.Source data

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