FIGURE 5.

Sp1 levels and specific DNA binding are substantially greater in orbital compared with dermal fibroblasts. A, nuclear protein from four orbital and two dermal fibroblast strains were subjected to Western immunoblot for Sp1 protein using a specific Ab against Sp1, as described under “Experimental Procedures.” Filters were re-probed with an Ab directed against β-actin. B, nuclear proteins were extracted from isolated nuclei of three orbital and two dermal fibroblast strains and subjected to EMSA using a ³²P-labeled oligonucleotide conforming to the consensus sequence of human Sp1 as a probe. Reactions were loaded onto native polyacrylamide gels, electrophoresed, and exposed to x-ray film. C, orbital fibroblast nuclear extract was subjected to EMSA using the ³²P-labeled consensus Sp1 binding sequence as a probe and competing the reaction with excess unlabeled Sp1 or a control (NF-κB) (100× molar excess each). D, nuclear protein extracts from orbital fibroblasts and HeLa cells were subjected to EMSA and “supershift” analysis using an anti-Sp1 monoclonal Ab. Studies were repeated at least three times.