Anti-LEF1 antibody [EP2030Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (AB53293)

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling LEF1 with purified ab53293 at 1/20 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (AB53293)

Paraffin-embedded human colon adenocarcinoma tissue stained for LEF1 using ab53293 (unpurified) at 1/50 dilution in immunohistochemical analysis.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (AB53293)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling LEF1 with purified ab53293 at 1/50 dilution (1.94 μg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (AB53293)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling LEF1 with purified ab53293 at 1/50 dilution (2.0 μg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (AB53293)

Intracellular flow cytometric analysis of permeabilized Jurkat cells using ab53293 (unpurified) (red) or a rabbit IgG (negative) (green).

• WB

Lab

Western blot - Anti-LEF1 antibody [EP2030Y] (AB53293)

False colour image of Western blot : Anti-LEF1 antibody [EP2030Y] staining at 1/5000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53293 was shown to bind specifically to LEF1. A band was observed at 40/53 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lef1 knockout cell line ab274898 (knockout cell lysate ab274956). To generate this image, wild-type and Lef1 knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent ab133456) and imaged with 8 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293) at 1/5000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 40 µg

Lane 2:

Lef1 knockout Jurkat cell lysate at 40 µg

Lane 2:

Western blot - Human LEF1 knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-lef1-knockout-jurkat-cell-line-ab274898'>ab274898</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Predicted band size: 44 kDa

Observed band size: 40 kDa

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• WB

Lab

Western blot - Anti-LEF1 antibody [EP2030Y] (AB53293)

This antibody recognizes all 7 LEF1 isoforms with the MW ranging from 23 to 44 KD based on immunogen blast.

All lanes:

Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293) at 1/5000 dilution

All lanes:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 44 kDa

Observed band size: 23-44 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (AB53293)

ab53293 (unpurified) staining LEF1 in RAMOS (human Burkitt's lymphoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain and the negative control was PBS only.

• WB

CiteAb

Western blot - Anti-LEF1 antibody [EP2030Y] (AB53293)

LEF1 western blot using anti-LEF1 antibody [EP2030Y] ab53293. Publication image and figure legend from Tang, L., Chen, Y., et al., 2020, J Cancer, PubMed 31897227.

ab53293 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 ab53293 please see the product overview.

DCST1-AS1 regulates the downstream proteins of miR-873-5p. (A) The expression level of IGF2BP1 in BRCA was analyzed by TCGA data. **, p < 0.0001. (B) TCGA data analysis showed that IGF2BP1 expressed higher levels in TNBC and luminal subtypes than normal controls, with significant differences. #, p < 0.05, *, p < 0.01. (C) The relationship between the expression of IGF2BP1 and the prognosis of BRCA patients was analyzed by TCGA data. P = 0.0054. (D) Western blot showed a decrease in the expression of IGF2BP1, MYC, LEF1 and CD44 in DCST1-AS1 interfering cells. (E) The miR-873-5p inhibitor was transfected into BT-549-si cells, and the expression of IGF2BP1 protein was measured by Western blot. (F) The miR-873-5p inhibitor was transfected into BT-549-si cells and cell proliferation was measured by CCK8 assay. *, p < 0.01. (G) The miR-873-5p inhibitor was transfected into BT-549-si cells and cell migration was measured by wound healing assay. *, p < 0.01.

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