重组Anti-ATG4A抗体[EPR4122]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG4A antibody [EPR4122] (AB108322)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue using ab108322 at 1/100 dilution.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG4A antibody [EPR4122] (AB108322)

Immunohistochemical analysis of paraffin-embedded Human brain tissue using ab108322 at 1/100 dilution.

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

• WB

Unknown

Western blot - Anti-ATG4A antibody [EPR4122] (AB108322)

All lanes:

Western blot - Anti-ATG4A antibody [EPR4122] (ab108322) at 1/1000 dilution

Lane 1:

Fetal liver tissue lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

Jurkat cell lysate at 10 µg

Lane 4:

Fetal brain tissue lysate at 10 µg

Secondary

All lanes:

Standard HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 45 kDa

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• WB

Lab

Western blot - Anti-ATG4A antibody [EPR4122] (AB108322)

Lanes 1 - 4 : Merged signal (red and green). Green - ab108322 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab108322 was shown to react with ATG4A in wild-type HeLa cells in Western blot with loss of signal observed in ATG4A knockout cell line ab265738 (ATG4A knockout cell lysate ab257846). Wild-type HeLa and ATG4A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab108322 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG4A antibody [EPR4122] (ab108322) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATG4A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATG4A knockout HeLa cell line (<a href='/products/cell-lines/human-atg4a-knockout-hela-cell-line-ab265738'>ab265738</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 45 kDa

Observed band size: 45 kDa

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• WB

CiteAb

Western blot - Anti-ATG4A antibody [EPR4122] (AB108322)

ATG4A western blot using anti-ATG4A antibody [EPR4122] ab108322. Publication image and figure legend from Zhu, K., Yuan, Y., et al., 2020, Aging (Albany NY), PubMed 32302291.

ab108322 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 ab108322 please see the product overview.

miR-22/miR-142 targets the key autophagy proteins ATG5, ATG4A and ULK1. (A) Predicted miR-142 binding sites in the ULK1, ATG4A, and ATG5 3'UTR. Wild-type and mutant-type ULK1, ATG4A, and ATG5 3'UTR reporter vectors containing wild-type or mutant-type miR-142 binding sites were constructed. (B–D) These vectors were co-transfected into HEK293 cells with miR-142 mimics or inhibitor, and the luciferase activity was determined. (E) Predicted miR-22 binding sites in ULK1. Wild-type and mutant-type ULK1 3'UTR reporter vectors containing wild- or mutant-type miR-22 binding sites were constructed. (F) These vectors were cotransfected into HEK293 cells with miR-22 mimics or inhibitor, and the luciferase activity was determined. (G, H) U2OS cells were transfected with miR-142 mimics in the presence or absence of Dox and examined for the protein levels of ULK1, ATG4A, and ATG5 were examined. (I, J) U2OS cells were transfected with miR-22 mimics in the presence or absence of Dox (5 μM), and the protein levels of ULK1 were examined. The data are presented as the mean ± SD of three independent experiments. *p<0.05, **p<0.01, compared to the control group; ##p<0.01, compared to the Dox group.

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