Anti-ATG3 antibody [EPR4801]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATG3 antibody [EPR4801] (AB108251)

Immunofluorescent staining of HeLa cells using ab108251 at 1/100.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATG3 antibody [EPR4801] (AB108251)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ATG3 (red) with ab108251 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG3 antibody [EPR4801] (AB108251)

ab108251, at a 1/100 dilution, staining Human ATG3 in muscle, using Immunohistochemistry, Formalin/PFA-fixed paraffin-embedded tissue.

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

• WB

Lab

Western blot - Anti-ATG3 antibody [EPR4801] (AB108251)

Lanes 1-4 : Merged signal (red and green). Green - ab108251 observed at 40 kDa. Red - loading control ab7291 observed at 50 kDa.

ab108251 Anti-ATG3 antibody [EPR4801] was shown to specifically react with ATG3 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266707 (knockout cell lysate ab257363) was used. Wild-type and ATG3 knockout samples were subjected to SDS-PAGE. ab108251 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ATG3 antibody [EPR4801] (ab108251) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

ATG3 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human ATG3 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-atg3-knockout-hek-293t-cell-line-ab266707'>ab266707</a>)

Lane 3:

K-562 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 35 kDa

Observed band size: 40 kDa

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• WB

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Western blot - Anti-ATG3 antibody [EPR4801] (AB108251)

All lanes:

Western blot - Anti-ATG3 antibody [EPR4801] (ab108251) at 1/10000 dilution

Lane 1:

K562 cell lysate at 10 µg

Lane 2:

HL-60 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit

Predicted band size: 35 kDa

Observed band size: 40 kDa

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• WB

CiteAb

Western blot - Anti-ATG3 antibody [EPR4801] (AB108251)

ATG3 western blot using anti-ATG3 antibody [EPR4801] ab108251. Publication image and figure legend from Agrotis, A., Pengo, N., et al., 2019, Autophagy, PubMed 30661429.

ab108251 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab108251 please see the product overview.

Additional loss of ATG4A in ATG4B KO cells only partially further reduces GABARAP subfamily priming and does not prevent rescue of autophagy by pre-primed LC3/GABARAP. (a) HeLa control, ATG4B KO and different ATG4A/B double knockout (DKO) clones were treated for 3 h with DMSO or 250 nM Torin1 + 10 nM baf A1 prior to lysis and western blot analysis. See also Figure S6A for analysis of the same clones by immunocytochemistry of endogenous GABARAPL1. (b) HeLa control, ATG4B KO and ATG4A/B DKO (c25) were transiently transfected with 3xFLAG-GABARAPL2-GFP. After 24 h, cells were treated for 3 h with DMSO or 250 nM Torin1 + 10 nM baf A1 prior to lysis and western blot. 3xFLAG-GABARAPL2-GFP was detected using anti-FLAG antibody. (c) HeLa cells stably transduced with GFP-GABARAPL1-G116 (Control and ATG4A/B DKO c25) and GFP-LC3B-G120 (ATG4A/B DKO c25) were treated for 3 h with DMSO or 250 nM Torin1 + 10 nM baf A1 prior to fixation and immunocytochemistry to reveal endogenous SQSTM1 and LAMP1 localization. Scale bar : 10 µm. See also Figure S6B for immunocytochemistry of untransduced cells. (d) Untransduced and stably-transduced HeLa control and ATG4A/B DKO (c25) cells were assessed for SQSTM1 protein level by western blot, 48 h after transfection with siRNA targeting RLUC as a negative control (lanes without +), or EGFP (lanes with +). GFP-LC3B-G120 and GFP-GABARAPL1-G116 were detected using anti-GFP antibody. (e) HeLa ATG4A/B DKO (c25) cells stably expressing GFP-LC3B-G120 were transfected with siRNA targeting RLUC (negative control), ATG3, ATG7 or GFP (positive control). After 48 h, cells were lysed and subject to western blot analysis to assess SQSTM1 protein level. GFP-LC3B-G120 was detected using anti-GFP antibody.

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