Anti-DMT1 antibody [4C6]

• WB

Lab

Western blot - Anti-DMT1 antibody [4C6] (AB55735)

Western blot : Mouse Monoclonal[4C6] to DMT1 ab55735 staining at 1/1000 dilution, shown in green; Rabbit anti GAPDH (ab181602) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 80 kDa in Wild-type A549 cell lysates with no signal observed at this size in SLC11A2 knockout A549 cell line (ab326025). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-DMT1 antibody [4C6] (ab55735) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human SLC11A2 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-slc11a2-knockout-a549-cell-line-ab326025'>ab326025</a>) at 20 µg

Secondary

All lanes:

Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

Predicted band size: 62 kDa

Observed band size: 80 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DMT1 antibody [4C6] (AB55735)

DMT1 antibody (ab55735) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human endometrium cancer.

This image was generated using the ascites version of the product.

• Flow Cyt

Unknown

Flow Cytometry - Anti-DMT1 antibody [4C6] (AB55735)

Overlay histogram showing SH-SY5Y cells stained with ab55735 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab55735, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol/permeabilized in 0.1% PBS-Tween used under the same conditions.

This image was generated using the ascites version of the product.

• IHC-Fr

AbReview36810****

Immunohistochemistry (Frozen sections) - Anti-DMT1 antibody [4C6] (AB55735)

IHC-Fr image of DMT1 staining on mouse duodenum using ab55735 (1 : 1000). The sections were fixed with paraformaldehyde and permeabilized using 0.1% TritonX in 0.1% PBS. The slides were blocked using 10 % donkey serum for 1 hour at 24°C. ab55735 was diluted 1 : 1000 using 0.1% TritonX with 0.1x PBS- 10% Donkeys and incubated with the slides at 4°C for 24 hours. The secondary antibody used was donkey polyclonal against mouse IgG conjugated to Alexa Fluor 568 (1 : 1000)

This image was generated using the ascites version of the product.

This image is courtesy of an abreview submitted by Dr. Ruma Raha-Chowdhury, Cambridge University, United Kingdom.

• WB

Unknown

Western blot - Anti-DMT1 antibody [4C6] (AB55735)

Western blot against tagged recombinant protein fragment (immunogen peptide) using ab55735 DMT1 antibody at 1ug/ml.

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-DMT1 antibody [4C6] (ab55735)

Predicted band size: 62 kDa

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• WB

CiteAb

Western blot - Anti-DMT1 antibody [4C6] (AB55735)

Western Blotting using Anti-DMT1 antibody [4C6], ab55735. 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, Representative western blots (top) of proteins involved in inflammation in MDM treated with LCC-12 and corresponding quantifications (bottom) (n = 8 donors). b, Immunoassay of cytokines secreted by MDM treated with LCC-12 during activation (n = 6 donors). c, PCA of RNA-seq comparing naMDM (n = 10 donors), aMDM (n = 10 donors) and MDM treated with LCC-12 during activation (n = 5 donors). d, Fluorescence microscopy quantifications of histone H3 methyl and acetyl marks in MDM treated with LCC-12. Quantifications were normalized against naMDM. At least 50 cells were quantified per donor per condition (n = 5–7 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 aMDM (n = 10 donors) and MDM treated with LCC-12 during activation (n = 5 donors). f, ChIP-seq tracks of selected genes involved in inflammation in MDM. g, Western blots of proteins involved in inflammation in aMDM under SOD2 knockdown conditions. h, Western blots of proteins involved in inflammation in aMDM under SLC25A3 knockdown conditions. i, Flow cytometry of wild-type (WT) and CD44 knockout (KO) aMDM. Gating strategy see Supplementary Information. j, Western blots of metal transporter proteins in WT and CD44-KO aMDM in n = 1 donor. k, Western blots of proteins involved in inflammation and histone marks in WT aMDM and CD44-KO aMDM for n = 3 donors. H3 is a sample processing control. For a and d Kruskal-Wallis test with Dunn’s post-test. For b, g and h 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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• WB

CiteAb

Western blot - Anti-DMT1 antibody [4C6] (AB55735)

Western Blotting using Anti-DMT1 antibody [4C6], ab55735. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

CD44 mediates the uptake of metals in inflammatory macrophages.a, Flow cytometry of cell surface markers in MDM. Monocytes treated as indicated to obtain pro-inflammatory or anti-inflammatory states. b, Flow cytometry of cell surface markers in MDM. Data representative of n = 13 donors. c, Bright field microscopy images of MDM. Scale bar, 20 µm. Morphological observations representative of n = 128 donors. d, ICP-MS of cellular metals in MDM (n = 9 donors). e, ICP-MS of cellular metals in aMDM under knockdown conditions of indicated genes (n = 6 donors). f, Western blots of cellular metal transporters in aMDM under knockdown conditions of indicated genes (n = 4 donors). g, Western blots of cellular metal transporters in aMDM under CD44 knockdown conditions (n = 6 donors). For d, f and g two-sided Mann-Whitney test. For e, Kruskal-Wallis test with Dunn’s post-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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• WB

CiteAb

Western blot - Anti-DMT1 antibody [4C6] (AB55735)

Western Blotting using Anti-DMT1 antibody [4C6], ab55735. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

CD44 mediates copper uptake.a, Experimental setup used to generate inflammatory monocyte-derived macrophages (MDMs). b, Flow cytometry of CD44 in MDMs. Data are representative of n = 13 donors. AU, arbitrary units. c, ICP-MS of cellular copper in MDMs (n = 9 donors). d, ICP-MS of cellular copper in aMDMs with short interfering RNA (siRNA) knockdown of indicated receptors and transporters (n = 6 donors). Copper transporter 1 (CTR1) is encoded by SLC31A1, CTR2 is encoded by SLC31A2, transferrin receptor 1 (TFR1) is encoded by TFRC, and divalent metal transporter 1 (DMT1) is encoded by SLC11A2. siCtrl, control siRNA. e, Representative western blots of metal transporters in MDMs (n = 7 donors). FC, fold change. f, ICP-MS of cellular copper in MDMs treated with anti-CD44 antibody RG7356 during activation (n = 7 donors). g, ICP-MS of cellular copper in MDMs treated with hyaluronate (0.6–1 MDa) (HA) or permethylated hyaluronate (meth–HA) during activation (n = 6 donors). h, Molecular structure of hyaluronate tetrasaccharide (top) and 1H NMR spectra (bottom) of copper–hyaluronate complexation experiment, recorded at 310 K in D2O. i, Fluorescence microscopy of a lysosomal copper(ii) probe (Lys-Cu) and FITC–hyaluronate in aMDMs treated with hyaluronidase (HD). At least 30 cells were quantified per donor (n = 6 donors). Scale bar, 10 µm. Rel., relative. c,e,f,i, Two-sided Mann–Whitney test. d,g, Kruskal–Wallis test with Dunn’s post test. In all box plots in the main figures, boxes represent the interquartile range, centre lines represent medians and whiskers indicate the minimum and maximum values. In graphs, each coloured dot represents an individual donor for a given panel.Source data

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• WB

CiteAb

Western blot - Anti-DMT1 antibody [4C6] (AB55735)

Western Blotting using Anti-DMT1 antibody [4C6], ab55735. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

CD44 mediates the uptake of metals in inflammatory macrophages.a, Flow cytometry of cell surface markers in MDM. Monocytes treated as indicated to obtain pro-inflammatory or anti-inflammatory states. b, Flow cytometry of cell surface markers in MDM. Data representative of n = 13 donors. c, Bright field microscopy images of MDM. Scale bar, 20 µm. Morphological observations representative of n = 128 donors. d, ICP-MS of cellular metals in MDM (n = 9 donors). e, ICP-MS of cellular metals in aMDM under knockdown conditions of indicated genes (n = 6 donors). f, Western blots of cellular metal transporters in aMDM under knockdown conditions of indicated genes (n = 4 donors). g, Western blots of cellular metal transporters in aMDM under CD44 knockdown conditions (n = 6 donors). For d, f and g two-sided Mann-Whitney test. For e, Kruskal-Wallis test with Dunn’s post-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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