All lanes : 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
Lane 2 : Nuclear extracts of Cal-1 cells infected with TCF4 shRNA

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated

Predicted band size: 71 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?


Exposure time: 40 seconds

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

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.

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).

Flow cytometric analysis of 1% paraformaldehyde-fixed, ice-cold methanol permeabilized 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.

 

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).

 

Anti-TCF-4 antibody [NCI-R159-6] (ab217668) at 1/200 dilution + SH-SY5Y (Human neuroblastoma cell line from bone marrow) nuclear extracts at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 71 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

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

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