Anti-FOXO3A antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-FOXO3A antibody (AB23683)

ICC/IF image of ab23683 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab23683, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% HepG2 fixed (10 min) HepG2 cells at 5µg/ml.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXO3A antibody (AB23683)

IHC image of FOXO3A staining in Human normal lung formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab23683, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-FOXO3A antibody (AB23683)

Lanes 1 - 4 : Merged signal (red and green). Green - ab23683 observed at 71 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab23683 was shown to recognize FOXO3A in wild-type HEK-293 cells as signal was lost at the expected MW in FOXO3 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and FOXO3 knockout samples were subjected to SDS-PAGE. ab23683 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FOXO3A antibody (ab23683) at 1 µg/mL

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human FOXO3 (FOXO3A) knockout HEK-293 cell lysate (<a href='/en-us/products/cell-lysates/human-foxo3-foxo3a-knockout-hek-293-cell-lysate-ab261649'>ab261649</a>) at 20 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Predicted band size: 71 kDa

Observed band size: 82 kDa

false

• WB

Unknown

Western blot - Anti-FOXO3A antibody (AB23683)

ab23683 detects a band at approximately 90 kDa that corresponds in size to that seen for FOXO3A. This protein has been shown to migrate at a size larger than the predicted molecular weight (see Yin et al., J. Biol. Chem., Vol. 279, Issue 44, 45721-45727). It also detects a band at 50 kDa which could be a cleavage fragment. Both bands are partially blocked by the addition of the immunizing peptide.

All lanes:

Western blot - Anti-FOXO3A antibody (ab23683) at 1 µg/mL

Lane 1:

Western blot - Jurkat whole cell lysate (<a href='/en-us/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 20 µg

Lane 2:

Western blot - Jurkat whole cell lysate (<a href='/en-us/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 20 µg with Human FOXO3A peptide (<a href='/en-us/products/unavailable/human-foxo3a-peptide-ab24031'>ab24031</a>)

Predicted band size: 71 kDa

Observed band size: 50 kDa,90 kDa

false

• WB

Lab

Western blot - Anti-FOXO3A antibody (AB23683)

Lanes 1 - 4 : Merged signal (red and green). Green - ab23683 observed at 82 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab23683 was shown to react with FOXO3A in wild-type cells in Western blot with loss of signal observed in FOXO3 knockout cell lines. Wild-type and FOXO3 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab23683 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at 1 μg/ml 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-FOXO3A antibody (ab23683) at 1 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human FOXO3 (FOXO3A) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-foxo3-foxo3a-knockout-hek-293t-cell-lysate-ab256922'>ab256922</a>) at 20 µg

Lane 3:

Wild-type HEK-293 cell lysate at 20 µg

Lane 4:

Western blot - Human FOXO3 (FOXO3A) knockout HEK-293 cell lysate (<a href='/en-us/products/cell-lysates/human-foxo3-foxo3a-knockout-hek-293-cell-lysate-ab261649'>ab261649</a>) at 20 µg

Predicted band size: 71 kDa

Observed band size: 82 kDa

false

• WB

Unknown

Western blot - Anti-FOXO3A antibody (AB23683)

ab23683 detects a band at approximately 90 kDa that corresponds in size to that seen for FOXO3A. This protein has been shown to migrate at a size larger than the predicted molecular weight (see Yin et al., J. Biol. Chem., Vol. 279, Issue 44, 45721-45727).

All lanes:

Western blot - Anti-FOXO3A antibody (ab23683) at 1 µg/mL

Lane 1:

Heart (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 2:

Western blot - Mouse skeletal muscle tissue lysate - total protein (<a href='/en-us/products/tissue-lysates/mouse-skeletal-muscle-tissue-lysate-total-protein-ab29711'>ab29711</a>) at 10 µg

Lane 3:

Heart (Rat) Tissue Lysate - normal tissue at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 71 kDa

Observed band size: 16 kDa,27 kDa,90 kDa

false

• WB

CiteAb

Western blot - Anti-FOXO3A antibody (AB23683)

FOXO3A western blot using anti-FOXO3A antibody ab23683. Publication image and figure legend from Gao, J., Liu, S., et al., 2018, Front Mol Neurosci, PubMed 30104959.

ab23683 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 ab23683 please see the product overview.

Mitochondrial respiratory chain and silent mating-type information regulation 2 homolog 3 (Sirt3) pathway mediated the protective effects of TLB in PC12 cells. Following pre-treatment with or without TLB for 1 h and incubated with 400 μM H2O2 for further 48 h, and the mitochondria in each group were isolated and purified. (A) Representative Western blots were shown for Sirt3, IDH2, forkheadboxO3a (FoxO3a)-, ac-SOD2, GPX-1 and uncoupling protein 2 (UCP2) proteins. (B) Quantitation of Sirt3 protein. (C) Quantitation of IDH2 protein. (D) Quantitation of Foxo3a protein. (E) Quantitation of ac-SOD2 protein. (F) Quantitation of GPX-1 protein. (G) Quantitation of UCP2 protein. (H) mRNA level of Sirt3 and mtDNA genes. (I) The activity of mitochondrial respiratory chain complex I. (J) The activity of adenosine triphosphate (ATP) (K) Nicotinamide adenine dinucleotide (NAD)+/NADH ratio. Data were presented as mean ± SD of three independent experiments. **p < 0.01 vs. untreated control cells; #p < 0.05, ##p < 0.01 vs. H2O2-treated cells.

false