Anti-ATG7 antibody [EP1759Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EP1759Y] (AB52472)

Immunocytochemistry/Immunofluorescence analysis of HT-29 (human colorectal adenocarcinoma) cells labelling ATG7 with purified ab52472 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue).

Secondary Only Control : PBS was used instead of the primary antibody as the negative control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EP1759Y] (AB52472)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling ATG7 with purified ab52472 at 1/100. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291 anti-Tubulin (mouse mAb) followed by ab150120, AlexaFluor^®594 goat anti-mouse secondary both at 1/1000. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used followed by anti-mouse secondary antibody (ab150120). For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATG7 antibody [EP1759Y] (AB52472)

Intracellular Flow Cytometry analysis of HeLa cells labelling ATG7 (red) with purified ab52472 at dilution of 1/100. The secondary antibody used was goat anti rabbit IgG (FITC) at 1/500. Cells were fixed with 4% paraformaldehyde. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG7 antibody [EP1759Y] (AB52472)

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma sections labelling ATG7 with purified ab52472 at a dilution of 1/500. The secondary antibody used was ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATG7 antibody [EP1759Y] (AB52472)

Overlay histogram showing HEK293 cells stained with unpurified ab52472 (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 (ab52472, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• IP

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Immunoprecipitation - Anti-ATG7 antibody [EP1759Y] (AB52472)

ab52472 at 1/30 dilution immunoprecipitating ATG7 in HEK293 whole cell lysate observed at 70 KDa (lanes 1 and 2).

Lane 1 (input) : HEK293 whole cell lysate 10ug

Lane 2 (+) : ab52472 + HEK293 whole cell lysate

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab52472 in HEK293 whole cell lysate

For western blotting, ab52472 was used followed by VeriBlot for IP Detection Reagent (HRP) (ab131366) for detection at a dilution of 1/10,000.

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

All lanes:

Immunoprecipitation - Anti-ATG7 antibody [EP1759Y] (ab52472)

Predicted band size: 49 kDa,77 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EP1759Y] (AB52472)

False colour image of Western blot : Anti-ATG7 antibody [EP1759Y] staining at 1/100000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52472 was shown to bind specifically to ATG7. A band was observed at 75 kDa in wild-type HeLa cell lysates with no signal observed at this size in ATG7 knockout cell line ab283307 (knockout cell lysate ab287353). To generate this image, wild-type and ATG7 knockout HeLa 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ATG7 antibody [EP1759Y] (ab52472) at 1/100000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATG7 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATG7 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-atg7-knockout-hela-cell-line-ab283307'>ab283307</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 77 kDa

Observed band size: 75 kDa

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• WB

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Western blot - Anti-ATG7 antibody [EP1759Y] (AB52472)

Blocking and Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-ATG7 antibody [EP1759Y] (ab52472) at 1/100000 dilution

Lane 1:

HEK293 whole cell lysate at 20 µg

Lane 2:

HepG2 whole cell lysate at 20 µg

Lane 3:

Jurkat whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 77 kDa

Observed band size: 70 kDa

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• WB

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Western blot - Anti-ATG7 antibody [EP1759Y] (AB52472)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : ATG7 knockout HAP1 cell lysate (20 μg)
Lane 3 : Jurkat cell lysate (20 μg)
Lane 4 : HepG2 cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab52472 observed at 77 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab52472 was shown to specifically react with ATG7 when ATG7 knockout samples were used. Wild-type and ProteinX knockout samples were subjected to SDS-PAGE. ab52472 and ab8245 (loading control to Apg7) were both diluted 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG7 antibody [EP1759Y] (ab52472)

Predicted band size: 77 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EP1759Y] (AB52472)

Western blot : Anti-ATG7 antibody [EP1759Y] ab52472 staining at 1/2000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 78 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in ATG7 knockout U-87 MG cell line. 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-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-ATG7 antibody [EP1759Y] (ab52472) at 1/2000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

ATG7 knockout U-87 MG at 20 µg

Lane 3:

THP-1 at 20 µg

Lane 4:

Jurkat at 20 µg

Secondary

All lanes:

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

Predicted band size: 78 kDa

Observed band size: 78 kDa

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• WB

CiteAb

Western blot - Anti-ATG7 antibody [EP1759Y] (AB52472)

Western Blotting using Anti-ATG7 antibody [EP1759Y], ab52472. Publication image from Rubinsztein, D. C. et al., 2016, Nat Commun, 26837467. Legend direct from paper.

Autophagy modulates Notch signalling pathway.(a) Representative western blot showing levels of Notch1 and its downstream effectors. Autophagy was inhibited in HEK cells by Smartpool siRNA (KD) for ATG7 or ATG16L1 or activated by pcDNA/Beclin-Flag transfection. Scr=scrambled siRNA. Actin is loading control. (b) Quantification of western blots for Notch1 and effectors, relative to actin, normalized for relevant control. *P<0.05 or **P<0.01 by paired t-test. n=3 in triplicates. Error bars=s.e.m. (c) Representative western blot showing effect of ATG7 Smartpool siRNA knockdown and ATG7-wt cDNA rescue on Notch1 levels. (d) Quantification of western blots for Notch1 and ATG7, relative to actin, normalized for relevant control. *P<0.05 or **P<0.01 by paired t-test. NS denotes not significant. n=4. Error bars=s.e.m. (e) Representative western blot showing the effect of ATG16L1 Smartpool siRNA knockdown and ATG16L1 mStr rescue on Notch1 levels. (f) Quantification of western blots for Notch1 and ATG16L1/ATG16L1 mStr, relative to actin, normalized for relevant control. *P<0.05 or **P<0.01 by paired t-test. n=3. Error bars=s.e.m. (g,h) HEK cells were transfected with control (scrambled) siRNA/ATG16L1 siRNA +Dll1 ligands (ATG16L1KD), or pcDNA/Beclin-Flag. Notch pathway activity was measured by firefly luciferase reporter assay with RBP-Jκ coupled reporter using Renilla luciferase with constitutive promoter as control. Ratio of sample to control is shown. *P<0.05 and **P<0.01. NS denotes not significant by unpaired t-test. n=3. Error bars=s.e.m. (i) Effect of autophagy on the nuclear translocation of NICD. Scale bar, 20 µm for images of the upper panel. Scale bar, 5 µm for all lower panel images. (j) HEK cells were treated with rapamycin/starved with HBSS, or transfected with pcDNA/Beclin-1. Magnifications of areas in white boxes are shown in lower images and with DAPI in lower panels. Scale bar, 10 µm for upper panels. Scale bar, 5 µm for bottom two rows.

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