重组Anti-FOXO3A抗体[EPR1950]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXO3A antibody [EPR1950] (AB109629)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling FOXO3A with unpurified ab109629 at 1/150 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• WB

Lab

Western blot - Anti-FOXO3A antibody [EPR1950] (AB109629)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109629 observed at 82 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab109629 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 ab109629 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-FOXO3A antibody [EPR1950] (ab109629) at 1/1000 dilution

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='/products/cell-lysates/human-foxo3-foxo3a-knockout-hek-293t-cell-lysate-ab256922'>ab256922</a>) at 20 µg

Lane 2:

Western blot - Human FOXO3 (FOXO3A) knockout HEK-293T cell line (<a href='/products/cell-lines/human-foxo3-foxo3a-knockout-hek-293t-cell-line-ab265069'>ab265069</a>)

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='/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

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• WB

Lab

Western blot - Anti-FOXO3A antibody [EPR1950] (AB109629)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109629 observed at 71 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109629 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. The membrane was blocked with 3% Milk. ab109629 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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 [EPR1950] (ab109629) at 1/1000 dilution

Lane 1:

Wild-type HEK 293 whole cell lysate at 20 µg

Lane 2:

FOXO3 knockout 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 line (<a href='/products/cell-lines/human-foxo3-foxo3a-knockout-hek-293-cell-line-ab260857'>ab260857</a>)

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

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• WB

Unknown

Western blot - Anti-FOXO3A antibody [EPR1950] (AB109629)

All lanes:

Western blot - Anti-FOXO3A antibody [EPR1950] (ab109629) at 1/1000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

SH-SY5Y cell lysate at 10 µg

Predicted band size: 71 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-FOXO3A antibody [EPR1950] (AB109629)

CUT&RUN profiling with FOXO3A antibody demonstrates robust genome-wide enrichment in wild-type (WT) cells, which is markedly diminished in FOXO3 knockout (KO) cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with FOXO3A antibody (Abcam ab109629, 0.5 µg). 500,000 HEK293T WT or KO (Abcam ab265069) cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramнrez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to FOXO3A WT, with red indicating high localized enrichment and blue denoting background. Validated antibodies show genome-wide enrichment above IgG background consistent with FOXO3A binding in WT cells and near complete loss of signal in KO cells.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-FOXO3A antibody [EPR1950] (AB109629)

CUT&RUN profiling with FOXO3A antibody reveals the expected genomic enrichment pattern in wild-type (WT) cells, which is substantially reduced in FOXO3 knockout (KO) cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with FOXO3A antibody (Abcam ab109629, 0.5 µg). 500,000 HEK293T WT or KO (Abcam ab265069) cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• WB

CiteAb

Western blot - Anti-FOXO3A antibody [EPR1950] (AB109629)

FOXO3A western blot using anti-FOXO3A antibody [EPR1950] ab109629. Publication image and figure legend from Li, L., Lin, L., et al., 2020, J Cell Mol Med, PubMed 31881122.

ab109629 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 ab109629 please see the product overview.

Activation of p65 mediates PUMA induction in response to gilteritinib treatment. A, HCT116 cells were treated with gilteritinib for 24 h at indicated concentration. Indicated proteins were analysed by Western blot. B, HCT116 cells were treated with 50 nmol/L gilteritinib at indicated time‐points. Expression of p‐p65 (S536) and p65 was analysed by Western blot. C, HCT116 cells were transfected with either a control scrambled siRNA or a p65 siRNA for 24 h and then treated with 50 nmol/L gilteritinib for 24 h. p65 and PUMA expressions were analysed by Western blot. D, HCT116 cells were treated with 10 μmol/L BAY11‐7082 for 1 h, and then with 50 nmol/L gilteritinib for 24 h. Nuclear fractions were isolated from cells and analysed for p65 expression by Western blot. Lamin A/C and β‐actin, which are expressed in nucleus and cytoplasm, respectively, were used as controls for loading and fractionation. E, HCT116 cells were treated with 10 μmol/L BAY11‐7082 for 1 h, and then with 50 nmol/L gilteritinib for 24 h. The level of p‐p65 (S536) and PUMA was analysed by Western blot. F, Chromatin immunoprecipitation (ChIP) was performed using anti‐p65 antibody on HCT116 cells following gilteritinib (50 nmol/L) treatment for 12 h. The IgG was used to control for antibody specificity. PCR was carried out using primers surrounding the p65 binding sites in the PUMA promoter

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