Figure 7
In vivo influence of PARP-1 activity on the expression and DNA binding of Sp1. (A) Nuclear proteins (5 μg) from PARP-1+/+ cells grown alone (-; lane 2) or in the presence of H2O2 (lane 3) or PJ34 (lane 4), added either individually or in combination (PJ34+ H2O2; lane 5), were incubated with the Sp1 labeled probe and formation of DNA/protein complexes monitored by EMSA on a 8% native polyacrylamide gel as in Figure 2. The position of both the Sp1 and Sp3 DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone (lane 1). (B) The extracts used in panel A were SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1 (sc-59), PARP (C-2-10) and PAR (10-H). The position of the appropriate molecular mass markers (60-, 120-, and 190 kDa) is indicated. (C) Nuclear proteins (5 μg) from primary cultures of HSKs grown for various periods of time (4-, 24- and 72 h) either alone (-; lanes 1, 4 and 7), or in the presence of H2O2 (lanes 2, 5 and 8) or both H2O2 and PJ34 (PJ34+ H2O2; lanes 3, 6 and 9), were incubated with the Sp1 labeled probe and formation of DNA/protein complexes monitored by EMSA on a 8% native polyacrylamide gel as in panel A. (D) The extracts used in panel C were analyzed by Western blotting with antibodies against Sp1 (sc-59), PARP (C-2-10) and β-actin (CLT9001). Densitometric analyses of the band intensities was determined for both the Sp1 and PARP-1 proteins and normalized to that measured for β-actin. Values are shown below each corresponding track.