Figure 4

Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1^+/+ and PARP-1^-/- cells. (A) Immunoprecipitation of the Sp1-protein complexes in PARP-1^+/+ and PARP-1^-/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1^+/+ and PARP-1^-/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. (B) Immunoprecipitation of the PARP-1-protein complexes in PARP-1^+/+ and PARP-1^-/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.