Figure 2

DNA binding properties of Sp1/Sp3 and NFI in PARP-1^+/+ and PARP-1^-/- cells. (A) EMSA analysis of Sp1/Sp3 and NFI. Crude nuclear proteins (5 μg) from both PARP-1^+/+ and PARP-1^-/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for either Sp1 (left) or NFI (right). Formation of DNA/protein complexes was then monitored by EMSA on an 8% (Sp1) and 10% (NFI) native polyacrylamide gel and their position revealed through autoradiography. The position of both the Sp1/Sp3 and NFI DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone. (B) Sp1 competition experiment in EMSA. The Sp1 labeled probe used in panel A was incubated with nuclear proteins (5 μg) from both PARP-1^+/+ and PARP-1^-/- cells in the presence of either no (-) or 100- and 500-fold molar excesses of unlabeled competitor oligonucleotides (either Sp1 or NFI). Formation of DNA/protein complexes was then monitored by EMSA on an 8% native gel. (C) NFI competition experiment in EMSA. Same as in panel B except that the NFI double-stranded oligonucleotide was 5'-end labeled and used as probe for the assay. (D) Supershift experiment in EMSA. Crude nuclear proteins from both PARP-1^+/+ and PARP-1^-/- cells were incubated with the either the Sp1 (5 μg proteins were used) or NFI (10 μg proteins were used) labeled probe in the presence of either no (-), or 2 μl of a polyclonal antibody directed against Sp1 (Sp1Ab) or Sp3 (Sp3Ab) and added either individually or in combination (Sp1+Sp3Ab) (left), or with a polyclonal antibody directed against NFI (right). Formation of both the Sp1/Sp3 and NFI complexes, as well as their corresponding supershifted complexes (SSC) is indicated. P: labeled probe alone; U: unbound fraction of the labeled probe.