FIG. 6.

Effect of Rec on the regulation of the endogenous c-myc gene by PLZF. (A) U937T cells that conditionally produce PLZF and a derived cell line which, in addition, expresses Rec from the CMV promoter/enhancer, give rise to equal levels of PLZF upon exposure to low levels of Tet. Western immunoblottings were performed on the indicated quantities of total cell protein with the anti-PLZF monoclonal antibody at a dilution of 1:50, the anti-β actin monoclonal antibody at a dilution of 1:2,500, and the anti-Rec antiserum K3086 at a dilution of 1:100. (B) Western blot analysis shows that Rec expression causes overproduction of endogenous c-Myc despite equally high levels of PLZF. The monoclonal c-Myc antibody 9E10 was used at a dilution of 1:500. The bar diagram indicates the relative signal intensities of the PLZF and c-Myc bands, normalized to β-actin and measured by laser densitometry. The standard deviations of the levels of c-Myc depending on Rec were determined from three blots. (C) Western blots documenting the elevated expression of c-Myc and of known c-Myc-responsive genes in the presence of Rec. The anti-p53 monoclonal antibody DO-1 was used at 1:2,000, the anti-PCNA monoclonal antibody SC56 was used at 1:200, the anti-IκBα antibody was used at 1:500, and the anti-actin antibody was used at 1:2,500. The bar diagrams show the signal intensities.