Anti-ALKBH1 antibody [EPR19215]

• IP

Supplier Data

Immunoprecipitation - Anti-ALKBH1 antibody [EPR19215] (AB195376)

ALKBH1 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate with ab195376 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab195376 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : K562 whole cell lysate 10μg (Input).

Lane 2 : ab195376 IP in K562 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab195376 in K562 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-ALKBH1 antibody [EPR19215] (ab195376)

Predicted band size: 43 kDa

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• WB

Supplier Data

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 10 µg

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 10 µg

Lane 4:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa

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Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

Human fetal kidney lysate at 10 µg

Lane 2:

Human colon lysate at 10 µg

Lane 3:

Human fetal liver lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Lanes 1 - 3 : Merged signal (red and green). Green - ab195376 observed at 43 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab195376 was shown to specifically react with in wild-type HAP1 cells as signal was lost in ALKBH1 knockout cells. Wild-type and ALKBH1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab195376 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

ALKBH1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A549 whole cell lysate at 20 µg

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) transfected with siRNA specifically targeting ALKBH1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 120s

• WB

Supplier Data

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-4 : 30 seconds Lane 5-7 : 3 minutes.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 5:

Mouse brain lysate at 10 µg

Lane 6:

Mouse spleen lysate at 10 µg

Lane 7:

Rat spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa

false

• WB

CiteAb

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Western Blotting using Anti-ALKBH1 antibody [EPR19215], ab195376. 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-ALKBH1 antibody [EPR19215] (AB195376)

Western Blotting using Anti-ALKBH1 antibody [EPR19215], ab195376. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

Mitochondrial copper(II) regulates epigenetic states and transcriptional programs of inflammatory macrophages.a, Quantitative mass-spectrometry-based proteomics of MDM (n = 8 donors). b, Representative western blots (top) of epigenetic modifiers identified by RNA-seq in aMDM and corresponding quantifications (bottom) (n = 6–8 donors). c, Genes encoding iron-dependent demethylases and acetyl-transferases found to be upregulated in aMDM are listed together with putative substrates and post-translational modifications (PTMs) products. d, Fluorescence microscopy quantifications of histone H3 methyl and acetyl marks in MDM. Quantifications represent aMDM normalized against naMDM. At least 50 cells were quantified per donor per condition (n = 5–11 donors). e, Scatter plot correlation of a representative donor of ChIP-seq reads count of histone marks in genes against RNA-seq of gene transcripts in MDM (n = 10 donors). f, ChIP-seq tracks of selected genes involved in inflammation in MDM. For b and d, two-sided Mann-Whitney test. Box plots : boxes represent interquartile range and median, and whiskers indicate the minimum and maximum values. Each colored dot represents a distinct donor for a given panel.Source data

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