Anti-FOXG1 抗体

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXG1 antibody (AB18259)

IHC image of FoxG1 staining in mouse brain E14 formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. 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 ab18259, 0.5 μg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Staining revealed in telencephalon (but not diencephalon) as expected.

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.

• IHC-P

AbReview4352****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXG1 antibody (AB18259)

ab18259 staining Foxg1 (1/50) in the telencephalon (but not the diencephalon) as expected. DAB-immunohistochemistry was performed on embryonic (E13.5) mouse brain (coronal paraffin-embedded sections) after microwave treatment with 10mM sodium citrate.

This image is courtesy of Vassiliki Fotaki, University of Edinburgh

• WB

Ap

Western blot - Anti-FOXG1 antibody (AB18259)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab18259 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-FOXG1 antibody (ab18259) at 1 µg/mL

Lane 1:

Human brain tissue lysate - total protein (ab29466) at 10 µg

Lane 2:

Brain (Mouse) Tissue Lysate at 10 µg

Lane 3:

E10 Mouse Embryo Brain Tissue Lysate at 10 µg

Lane 4:

Brain (Rat) Tissue Lysate at 10 µg

Lane 5:

Human spleen tissue lysate - total protein (negative control) at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (HRP) at 1/50000 dilution

Predicted band size: 52 kDa

Observed band size: 155 kDa,50 kDa,52 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-FOXG1 antibody (AB18259)

ab18259 detects a band of ~50kDa in brain lysates. This band is blocked by addition of the immunizing peptide ab19644 which suggests that is specific for FOXG1.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab18259 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody (ab97051), and visualised using ECL development solution.

All lanes:

Western blot - Anti-FOXG1 antibody (ab18259) at 1 µg/mL

Lane 1:

Human brain tissue lysate - total protein (ab29466) at 10 µg

Lane 2:

Brain (Mouse) Tissue Lysate at 10 µg

Lane 3:

Brain (Rat) Tissue Lysate at 10 µg

Lane 4:

Human brain tissue lysate - total protein (ab29466) at 10 µg with Human FOXG1 peptide (ab19644)

Lane 5:

Brain (Mouse) Tissue Lysate at 10 µg with Human FOXG1 peptide (ab19644)

Lane 6:

Brain (Rat) Tissue Lysate at 10 µg with Human FOXG1 peptide (ab19644)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 52 kDa

Observed band size: 50 kDa

true

Exposure time: 1min

• IHC

CiteAb

Immunohistochemistry - Anti-FOXG1 antibody (AB18259)

Immunohistochemistry using Anti-FOXG1 antibody, ab18259. Publication image from Rossi, S. et al., 2013, Nat Commun, 24356439. Legend direct from paper.

Elevated FOXG1 expression in GBM correlates with poor prognosis(a) Body-wide expression of FOXG1 across the GeneSapiens database, with each dot representing expression in a single sample. Anatomical sources of examined samples are indicated with coloured bars below the gene plot, with corresponding legends also shown. Anatomical samples with higher than average FOXG1 expression or an outlier expression profile are shown coloured. Vertical red arrow points to FOXG1 expression in glioma. (b) FFPE sections from normal brain or glioma of increasing grade were subjected to immunohistochemistry with a validated anti-FOXG1 antibody, followed by counterstaining with hematoxylin. Boxed areas in the top row define regions shown at higher magnification in the bottom row. A representative image is shown for each group. Scale bars : top row, 100 µm; bottom row, 50 µm. (c) Quantification of the number of FOXG1-positive nuclei in normal brain or glioma specimens. Data are represented as mean±SEM (grade II glioma, ns, not significant, n=12; grade III glioma, P=1.2x10−3, n=16; GBM, P<1.0x10−4, n=16; normal brain, n=13; ANOVA). (d) Western blot analysis of FOXG1 expression in normal brain and GBM tissue extracts. Expression of GAPDH is shown as loading control. Molecular size markers are indicated in kDa. One representative western blot result is shown (n≥3). (e) Kaplan-Meier curves comparing survival among GBM patients. Survival curves are shown for patients with tumours exhibiting either high (blue curve) or low (orange curve) FOXG1 mRNA levels compared to all GBM patients (grey curve). Data were obtained from the Rembrandt Database of the National Cancer Institute. Statistical analysis (P value calculated using Mantel-Cox test) for high vs all, or low vs all, FOXG1 levels is shown next to the corresponding curves.

• WB

CiteAb

Western blot - Anti-FOXG1 antibody (AB18259)

Western Blotting using Anti-FOXG1 antibody, ab18259. Publication image from Rossi, S. et al., 2013, Nat Commun, 24356439. Legend direct from paper.

FOXG1 silencing promotes expression of astroglial genes in BTICs(a) Western blot analysis of the indicated proteins in BTIC line BT025 cultured under non-differentiative or differentiative conditions. β-ACTIN is shown as loading control. Molecular size markers are indicated in kDa. One representative western blot result is shown in each case (n≥3). (b) Western blot analysis of the indicated proteins in non-silenced or FOXG1-silenced BT025 and BT048 cells (cultured under non-differentiative conditions). (c) ChIP analysis of SOX2 and BMI1 promoter occupancy in BT025 and BT048 cells using rabbit anti-FOXG1 antibody or rabbit pre-immune immunoglobulin. Input genomic DNA (Input) was also subjected to PCR. DNA size standards are indicated in base pairs. One representative ChIP experiment is shown in each case (n≥3). (d) Western blot analysis of the expression of the indicated proteins in BT048 cells (cultured under non-differentiative conditions) after transduction with lentiviral vectors expressing non-silencing shRNA or FOXG1 shRNA #1. GLUL, GLUTAMINE SYNTHETASE. (e) Quantification of Western blot data from separate experiments performed in BT048 cells, similar to the one shown in (d). Data are represented as mean±SEM (GFAP, P=8.0x10−3; S100β, P=2.4x10−5; GLUL, P=1.8x10−3; n=3; t-test). (f) qRT-PCR analysis of the indicated genes in BT048 cells after transduction with lentiviral vectors expressing non-silencing shRNA or FOXG1 shRNA #1. Values are expressed as fold change of FOXG1 shRNA-transduced cells over non-silencing shRNA-transduced cells using β-ACTIN as control (mean±SEM; GFAP, P=6.7x10−3; S100β, P=1.7x10−2; GLUL, P=2.7x10−3; n=3; ANOVA). (g) ChIP analysis of GFAP, S100β, and GLUTAMINE SYNTHETASE promoter occupancy in BT025 and BT048 cells performed using rabbit anti-FOXG1 antibody or control rabbit immunoglobulin, followed by PCR with primers specific for each of these genes. Input genomic DNA (Input) was subjected to PCR with the same primers.

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• WB

CiteAb

Western blot - Anti-FOXG1 antibody (AB18259)

Western Blotting using Anti-FOXG1 antibody, ab18259. Publication image from Rossi, S. et al., 2013, Nat Commun, 24356439. Legend direct from paper.

FOXG1 silencing impairs proliferation in cultured BTICs(a) Western blot analysis of FOXG1 in BTIC lines BT025 and BT048 after transduction with lentivirus expressing non-silencing shRNA or FOXG1 shRNA #1 and #2 reagents. β-ACTIN is shown as loading control. Molecular size markers are indicated in kDa. One representative western blot result is shown in each case (n≥3). (b) Quantification of the sphere-forming ability of non-silenced or FOXG1-silenced BT025 and BT048 cells passaged for two generations (mean±SEM; BT025 : shRNA #1, primary spheres, P=2.0x10−4, n=10; secondary spheres, P=2.3x10−3, n=6; shRNA #2, primary spheres, P=9.1x10−3, n=3; secondary spheres, P=5.5x10−3, n=3; BT048 : shRNA #1, primary spheres, P=6.5x10−3, n=5; secondary spheres, P=9.8x10−3, n=5; shRNA #2, primary spheres, P=8.0x10−3, n=6; secondary spheres, P=2.7x10−2, n=4; ANOVA). (c) Quantification of the percent of FOXG1-silenced BT025 and BT048 cells that incorporated BrdU compared to non-silenced cells (mean±SEM; BT025 : shRNA #1, P=2.2x10−3, n=7; shRNA #2, P=1.7x10−3, n=4; BT048, shRNA #1, P=1.1x10−3, n=5; shRNA #2, P=3.0x10−4, n=4; ANOVA). (d) Western blot analysis of PCNA and p21Cip1 (p21) expression in non-silenced or FOXG1-silenced BT025 and BT048 cells. (e) Senescence-associated β-GALACTOSIDASE activity (pH : 6.0) in non-silenced or FOXG1-silenced BT025 cells. Top row, GFP fluorescence; bottom row, 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal) staining. Scale bar : 20 µm. (f) Quantification of the percent of FOXG1-silenced BT025 and BT048 cells positive for X-Gal staining compared to non-silenced cells (mean±SEM; BT025 : shRNA #1, P=6.1x10−3, n=5; shRNA #2, P=8.5x10−3, n=4; BT048 : shRNA #1, P=2.7x10−2, n=4; shRNA #2, P=4.0x10−2, n=4; ANOVA). (g) qRT-PCR analysis of the indicated genes in non-silenced or FOXG1-silenced BT048 cells (mean±SEM; p21Cip1, P=7.0x10−4; β-GALACTOSIDASE (β-Gal), P=1.3x10−2; GADD45A, P<1.0x10−4; n=3; ANOVA). (h) ChIP analysis of p21Cip1, β-GALACTOSIDASE, and GADD45A promoter occupancy in BT025 and BT048 cells performed using rabbit anti-FOXG1 antibody or control rabbit immunoglobulin. Input genomic DNA (Input) was also subjected to PCR. DNA size standards are indicated in base pairs. One representative ChIP experiment is shown in each case (n≥3).

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