spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 5


Fig. 5. Endogenous RUNX1/AML1 and AML1-ETO interact with UBF1 on the rDNA repeats. (A) Immunoprecipitation analysis was carried out with an antibody for UBF1 followed by western blotting with AML1-ETO- and RUNX1/AML1-specific antibodies. Both RUNX1 and AML1-ETO are detected in western blot analysis, however AML1-ETO shows greater interaction with UBF1 when compared with RUNX1/AML1. (B) Endogenous RUNX1/AML1 and AML1-ETO were immunoprecipitated from Kasumi-1 cells using rabbit polyclonal antibodies that specifically recognize the C-terminus of RUNX1/AML1 or the ETO moiety. A mouse monoclonal antibody was used to detect endogenous UBF1 by immunoblotting. UBF1 and IgG heavy chain are indicated. (C) Immunofluorescence microscopy was performed in Kasumi-1 cells to detect endogenous RUNX1/AML1 (green), UBF1 (red) or AML1-ETO (green) with DAPI staining (blue). There is colocalization, albeit limited, of both RUNX1/AML1 and AML1-ETO with nucleolar UBF1 during interphase. (D) Electrophoretic mobility shift assays were performed with a human rDNA probe spanning a RUNX-binding element and nuclear extracts from Kasumi-1 cells. Competition assays with 100-fold molar excess of unlabeled wild-type, mutant or RUNX consensus oligonucleotide were performed to establish the specific protein-DNA complex (left) as indicated. Super-shift immunoassays were performed by incubating binding reactions with the indicated antibodies (right). Normal IgG was used as a negative control (control). The arrow on the right indicates the supershift band. (E) ChIP-reChIP assays with endogenous proteins in interphase Kasumi-1 cells using UBF1 antibody (primary ChIP) and second immunoprecipitation (reChIP) with antibodies directed against UBF1, RUNX1/AML1, AML1-ETO or IgG. The re-ChIP data are plotted as a percentage immunoprecipitation of the primary ChIP (set as 100%). Each of the regions was immunoprecipitated with similar efficiency in the primary ChIP. These results show that RUNX1/AML1 and AML1-ETO each can co-occupy rDNA repeats with UBF1.





Right arrow Return to article