Figure 2.

EMSA of tissue factor promoter DNA-binding motifs: effect of hypoxia. (A) EMSA using R2 from the tissue factor promoter. Rat mononuclear phagocytes (≈10⁷, A) were exposed to normoxia (N) or hypoxia (H; pO₂ ≈ 12–14 torr) for 45 min, nuclear extracts were prepared, and EMSA was performed using ³²P-labeled oligonucleotide for R2 (−96/−66 bp; ref. 9). Each lane received 10 μg/lane of total nuclear extract protein. The arrows indicate bands corresponding to Egr and Sp1 (A). Note that the unlabeled lower band observed in lanes 2, 3, and 7 may represent Sp3 (38). A 100-fold excess of the indicated unlabeled (cold) oligonucleotide with a consensus sequence of Sp1 or Egr was added. (B) EMSA using consensus oligonucleotide probe Egr. Nuclear extracts were harvested from normoxic/hypoxic HeLa cells and EMSA was performed with the indicated ³²P-labeled oligonucleotide probe. Excess unlabeled Sp1, AP-1, or Egr oligonucleotide was added to certain lanes, and either rabbit anti-Egr-1 IgG (1:5 and 1:50 dilution) or nonimmune IgG (1:5) was added to other lanes. (C) EMSA using consensus oligonucleotide for Egr, as above, and nuclear extracts from normoxic/hypoxic murine lung. Mice were subjected to hypoxia (≈6% oxygen; ref. 6) for 45 min, lungs were rapidly harvested, and nuclear extracts were prepared. EMSA was performed as above, and, where indicated, anti-Egr-1 IgG or nonimmune IgG (Santa Cruz Biotechnology) was added. (D) EMSA using consensus oligonucleotide for Sp-1 and nuclear extracts from normoxic/hypoxic murine lung was performed using the same conditions as above (+/+, wild-type mice; −/−, Egr-1 null mice). Results shown are representative of a minimum of four experiments.