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. 2002 Dec 1;30(23):5261–5268. doi: 10.1093/nar/gkf658

Figure 1.

Figure 1

Figure 1

Figure 1

(A) Skip and c-Ski synergistically overcome pRb-mediated repression of the AdE2 promoter. pRb null Saos-2 cells were transfected with 3 µg of the AdE2 CAT reporter construct together with 0.1 µg of pRb expression plasmid as indicated. Effects of Skip and c-Ski were assessed by transfecting them in increasing amounts either alone or in combination as shown (concentrations are in µg). After 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (B) Skip and c-Ski together inhibits transcriptional repression by pRb. A mixture of Gal4 site containing TK CAT reporter, the Gal4-Rb and the plasmid expressing Skip and c-Ski were transfected into U2OS cells as indicated, and after 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (C) Analysis of pRb expression in the presence of ectopic Skip and Ski expression. U2OS cells were transfected with the plasmids pCMV-Rb (0.1 µg), pCDNA3-HA-Skip/pCMV-Ski (0.5, 1 and 2 µg of each as shown) and pCMV-βGal (0.1 µg). Cell extracts were analyzed by western blotting for Rb (αRb), Skip (αHA), βGal (α-βGal). The Coomassie protein stain of an SDS–PAGE run in parallel with an aliquot of the extract used for the western blot is shown in the bottom panel and the numbers indicate the position of molecular weight markers.