FIG. 6.

(A) Western blot comparison of the viral multiplication in PDA cultures of two different strains of JCV. Blots utilized nuclear extracts from PDA cultures that had been transfected with either Mad-1 (pM1TC) or Archetype (pJC-CY) plasmids, resolved on 4 to 12% gradient gels, transferred to a PVDF membrane, and probed with anti-Vp-1 and anti-β-actin. The anti-β-actin blot serves as the total protein loading control. (B) An ATPA utilizing Western blot comparison of Smad binding profiles derived from either the JCV Mad-1 (ATP-Mad-1) or Archetype (ATP-Arche) promoter nucleotide sequences. This ATPA utilized nuclear extracts from PDA cultures that were either untreated (−) or treated with 5 ng/ml of TGF-β1 (+). Proteins from these nuclear extracts that bound to ATP-Mad-1 or ATP-Arche were eluted by boiling into protein loading buffer, resolved on 4 to 12% gradient gels, transferred to a PVDF membrane, and probed with anti-Smad2 and anti-Smad4. A nuclear extract from TGF-β1-treated PDA that was not subjected to binding was also loaded on the gels as an unbound control. Results included are representative of three independent experiments.