Figure 1.

Rb/p130-mediated repression independent of histone deacetylase recruitment. (A) Schematic representation of the pSVECG reporter. Constitutive CAT expression is driven by the SV40 promoter/enhancer. Upstream Gal4 sites provide a binding site for Gal4 DNA biding domain fusion proteins. (B) C33A cells were transiently transfected with 1 μg of β-gal, 0.5 μg of the pSVECG reporter, and 2 μg of Gal4-Rb, Gal4-RbΔp34, Gal4-p130, or Gal4-p130^C894F or 5 μg of Gal4-Rb^C706F. Western blotting was performed to verify that transfected constructs expressed equal protein. For the repression assay using the pocket domain, 0.5 μg of pSVECG was transfected with 3 μg of Gal4-p130 pocket. As controls, C33A cells were transfected with 1 μg of β-gal, 0.5 μg of pSVECG reporter, and 2 or 3 μg of a vector encoding a Gal4 DNA binding domain (control vector). Cells were harvested 40 hours posttransfection, and CAT activity was assayed. β-gal values were used to normalize for transfection efficiency. Results of typical experiments are shown.