Anti-FTO antibody [EPR6894]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FTO antibody [EPR6894] (AB126605)

Immunohistochemical staining of paraffin embedded human hepatocellular carcinoma with purified ab126605 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FTO antibody [EPR6894] (AB126605)

Immunofluorescence staining of BxPC-3 cells with purified ab126605 at a working dilution of 1 in 250, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 555 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab126605 was used at a dilution of 1/200 followed by an Alexa Fluor^® 488 goat anti-mouse antibody at a dilution of 1/500.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FTO antibody [EPR6894] (AB126605)

Immunohistochemical staining of FTO in paraffin embedded human adrenal gland tissue using unpurified ab126605 at a 1/50 dilution.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-FTO antibody [EPR6894] (AB126605)

Unpurified ab126605 at a 1/50 dilution staining FTO in BxPC3 cells by immunofluorescence.

• WB

Lab

Western blot - Anti-FTO antibody [EPR6894] (AB126605)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-FTO antibody [EPR6894] (ab126605) at 1/10000 dilution

Lane 1:

Molt-4 cell lysate at 20 µg

Lane 2:

HEK293 cell lysate at 20 µg

Lane 3:

BxPC-3 cell lysate at 20 µg

Lane 4:

Caco-2 cell lysate at 20 µg

Lane 5:

SH-SY5Y cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

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• WB

Lab

Western blot - Anti-FTO antibody [EPR6894] (AB126605)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : FTO knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HEK293 whole cell lysate (20 μg)
Lane 4 : MOLT4 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab126605 observed at 58 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab126605 was shown to specifically react with FTO in wild-type HAP1 cells. No band was observed when FTO knockout samples were examined. Wild-type and FTO knockout samples were subjected to SDS-PAGE. ab126605 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FTO antibody [EPR6894] (ab126605)

Predicted band size: 58 kDa

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• WB

Unknown

Western blot - Anti-FTO antibody [EPR6894] (AB126605)

All lanes:

Western blot - Anti-FTO antibody [EPR6894] (ab126605) at 1/1000 dilution

Lane 1:

MOLT4 cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

SH SY5Y cell lysate at 10 µg

Lane 4:

BxPC3 cell lysate at 10 µg

Lane 5:

Caco 2 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

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• WB

Supplier Data

Western blot - Anti-FTO antibody [EPR6894] (AB126605)

False colour image of Western blot : Anti-FTO antibody [EPR6894] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab126605 was shown to bind specifically to FTO. A band was observed at 58 kDa in wild-type MCF7 cell lysates with no signal observed at this size in FTO knockout cell line. To generate this image, wild-type and FTO knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-FTO antibody [EPR6894] (ab126605) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human FTO knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-fto-knockout-mcf7-cell-line-ab282631'>ab282631</a>)

Lane 2:

FTO knockout MCF7 cell lysate at 20 µg

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

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• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-FTO antibody [EPR6894] (AB126605)

Immunocytochemistry-immunofluorescence using Anti-FTO antibody [EPR6894], ab126605. Publication image from Koh, C. W. Q. et al., 2019, Nat Commun, 31822664. Legend direct from paper.

FTO overexpression causes aberrant mRNA methylation-suppression in the nucleus.a Immunofluorescence images of WT versus Fto-KO cells. Anti-FTO i and anti-FTO ii antibodies are respectively Abcam ab126605 and Santa Cruz sc271713. Anti-FTO i is specific for endogenous FTO while anti-FTO ii is not. Scale bar denotes 10 µm. b Immunofluorescence images of C-terminal-3 x -FLAG-tagged-WT-FTO in HEK293T. Scale bar denotes 10 µm. c, d m6ACE (green) overlaid on Input (orange) read-start RPM counts mapped to representative mRNAs. Sequence corresponds to the same strand as the m6A site. Blue horizontal bars represent transcript models. e Scatterplot of average RML of WT versus WT-Fto-OE cells. WT-FTO-affected are sites with RML reduction of at least log2fold of 2 (Student’s t-test p < 0.05) in WT-Fto-OE compared to WT cells. f MEME analysis of the sequence contexts of FTO-affected sites. g Model for cytoplasmic RNA methylation-reversal. h Model for disruptive methylation suppression by RNA demethylases. (Left panel) Most methylated RNAs (blue) are not demethylated by demethylases but simply undergo eventual RNA decay. (Middle panel) Selected RNAs (green) that ought to remain unmethylated are acted on almost simultaneously by both methyltransferase and demethylase, resulting in no net accumulation of RNA methylation. (Right panel) In the absence of demethylases, RNA methylation accumulates anomalously, which disrupts regular RNA processing.