Anti-TCF-4 antibody [NCI-R159-6]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Immunohistochemical analysis of 4% Formalin fixed human tonsil labeling TCF-4 with ab217668 at 1/100 dilution. Universal DAB Detection Kit was used for detection of IHC staining on an automated system.

pDCs : plasmacytoid dendritic cells.

GC : Germinal Center.

The data was provided by our collaborator Dr. Louis M. Staudt, and published in Cancer Cell 30, 764-778, 2016 (PMID : 27846392).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Flow cytometry overlay histogram showing SH-SY5Y cells stained with ab217668 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in nan followed by the antibody (ab217668) (1x 10⁶in 100μl at 0.1μg/ml (1/19400)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in SH-SY5Y Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Intracellular flow cytometric analysis of 1% paraformaldehyde-fixed, ice-cold methanolpermeabilized Cal-1 cells (Human plasmacytoid dendritic cell line) (black - positive control) and Cal-1 cells infected with either Ctrl (left green) or TCF-4 (right green) shRNA, labeling TCF-4 with ab217668 at 1/100 dilution (green and black) compared with a Rabbit IgG control (grey). Goat anti-Rabbit IgG (Alexa Fluor^® 647) at 1/500 dilution was used as the secondary antibody.

The data was provided by our collaborator Dr. Louis M. Staudt, and published in Cancer Cell 30, 764-778, 2016 (PMID : 27846392). Several TCF4 shRNAs were used. This FC image shows data for shRNA TCF4

1.

• ChIP

Supplier Data

ChIP - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Cal-1 cells (Human plasmacytoid dendritic cell line) were cross-linked with 1% formaldehyde for 5 min at RT. Cross-linked cells were first washed with ice-cold PBS and then resuspended in ice-cold RIPA buffer (10mM Tris-HCl pH8, 140 mM NaCl, 1mM EDTA pH 8, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS and 0.1% Sodium Deoxycholate) to a final concentration of 5x10⁶ cells/ml. DNA was sheared with a Misonix XL sonicator, by performing 12 x 45'' sonication cycles at power setting of 5. For each ChIP reaction, 2x10⁷ chromatin cell equivalents were incubated overnight with10 μg of ab217668. The following day, chromatin/antibody complexes were incubated with 50 μl of Protein G/Protein A magnetic beads mix (G to A ratio 3 : 1) for 4 h at 4°C. Normal rabbit IgG was added to the beads as control.

The TCF-4 locus ChIP-seq tracks for BRD4 (blue), RNA Pol2 (red), and TCF-4 (green) are shown for Cal-1 cells. This data was kindly provided by our collaborator Dr. Louis M. Staudt, and has been published (PMID : 27846392).

• WB

Supplier Data

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

The data was provided by the collaborator Dr. Louis M. Staudt, NCI, NIH.

All lanes:

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (ab217668) at 1/10000 dilution

Lane 1:

Nuclear extracts of Cal-1 cells (Human plasmacytoid dendritic cell line) infected with control TCF4 shRNA at 20 µg

Lane 2:

Nuclear extracts of Cal-1 cells infected with TCF4 shRNA at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated

Predicted band size: 71 kDa

Observed band size: 90 kDa

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Blocking and Diluting buffer and concentration : 5% NFDM /TBST

The isoforms expression pattern is consistent with the literatures (PMID : 21789225).

All lanes:

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (ab217668) at 1/200 dilution

All lanes:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) nuclear extracts at 10 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 1/100000 dilution

Predicted band size: 71 kDa

Observed band size: 90 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Blocking/Dilution buffer : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (ab217668) at 1/1000 dilution

Lane 1:

Daudi (human Burkitt's lymphoma lymphoblast) cytoplasmic fraction at 20 µg

Lane 2:

Daudi nuclear fraction at 20 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 1/100000 dilution

Predicted band size: 71 kDa

Observed band size: 58 kDa,79 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Blocking/Dilution buffer : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-TCF-4 antibody [NCI-R159-6] (ab217668) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 71 kDa

Observed band size: 58 kDa,79 kDa

true

Exposure time: 3min

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TCF-4 antibody [NCI-R159-6] (AB217668)

Immunohistochemical analysis of 4% Formalin fixed Blastic plasmacytoid dendritic cell neoplasm (BPDCN) cell pellets after selection and induction of shRNA expression for 1 day, labeling TCF-4 with ab217668 at 1/100 dilution. Universal DAB Detection Kit was used for detection of IHC staining on an automated system.

The data was provided by our collaborator Dr. Louis M. Staudt, and published in Cancer Cell 30, 764-778, 2016 (PMID : 27846392). Several TCF-4 shRNAs were used. This IHC image shows data for shRNA TCF4 #2.