Sp1, Sp3, USF-1, and NF-YA levels in CMK and CMS nuclear extracts and the role of Sp1/Sp3 phosphorylation of in vitro binding to the CBS −1b promoter.

(A) Fifty micrograms of nuclear extract proteins from CMS, CMK, and HepG2 (control) cells were fractionated on 7.5% (Sp1, Sp3) or 12% (USF1, NF-YA) polyacrylamide gels with SDS and electroblotted onto PVDF membranes. Immunoreactive Sp1, Sp3, USF-1, and NF-YA proteins were detected with Sp1, Sp3, USF-1, and NF-YA antibodies and Lumi-Light Western blotting substrate. Comparable protein levels of the transcription factors were found for both the CMK and CMS cells. (B) Nuclear extracts from CMK cells were pretreated with calf intestinal alkaline phosphatase (CIP), ³²P-labeled FPD probe was added, and following incubation on ice for 30 minutes, the DNA protein complexes were electrophoresed, as described in “Materials and methods.” Ten micrograms of the treated nuclear extracts were used for the gel shift assays. Decreased binding of Sp1/Sp3 to the³²P-labeled FPD probe was observed following CIP treatment. NA indicates no addition.