Fig. 4.
Fig. 4. Binding of HIF-1 to the rat PAI-1 promoter region. (A) Oligonucleotides: the HIF-1 consensus sequence BACGTSSK32were B=G/C/T, S=G/C, and K=G/T and the sense strand of the rat PAI-1 promoter sequence -177/-152 containing the HRE-1 and HRE-2 site, the sense strands of the oligonucleotides containing HRE-1, HRE-2, and the oligonucleotide with a mutation in the HRE-2 site M-HRE are shown. Bases matching the consensus sequences are underlined, mutated bases are in lower case letters. (B) EMSA, left panel: the32P-labeled PAI-1 HRE-1/2 oligonucleotide was incubated with either 10 μg protein of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells in the absence or presence of preimmune serum or rat HIF-1 antibody as indicated (cf, Materials and Methods). In EMSA with antibodies, the nuclear extracts were preincubated with 1 μL of the HIF-1 antibody for 2 hours at 4°C before adding the labeled probe. Middle panel: the32P-labeled HRE-2 (-168/-152) oligonucleotide and the PAI-1 HRE-1 (-182/-166) oligonucleotide were incubated with either 10 μg of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells in the absence or presence of preimmune serum or rat HIF-1 antibody as indicated. The inducible (I) HIF-1 complex was seen with the HRE-2, but not with the HRE-1 oligonucleotide. Right panel: the 32P-labeled mutated HRE-2 (-168/-152) oligonucleotide (M-HRE) was incubated with either 10 μg of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells or with extracts from hypoxic cells in the presence of rat HIF-1 antibody as indicated. The image had to be overexposed to visualize the weak constitutive binding. The DNA-protein binding was analyzed by electrophoresis on 5% native polyacrylamide gels. I, induced HIF-1 complex; C, constitutive complex; S, supershifted complex. (C) Western analysis of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells. A total of 20 μg nuclear extract from 2 different preparations was resolved on a 10% SDS gel and probed with preimmune serum or the HIF-1 antibody.

Binding of HIF-1 to the rat PAI-1 promoter region. (A) Oligonucleotides: the HIF-1 consensus sequence BACGTSSK32were B=G/C/T, S=G/C, and K=G/T and the sense strand of the rat PAI-1 promoter sequence -177/-152 containing the HRE-1 and HRE-2 site, the sense strands of the oligonucleotides containing HRE-1, HRE-2, and the oligonucleotide with a mutation in the HRE-2 site M-HRE are shown. Bases matching the consensus sequences are underlined, mutated bases are in lower case letters. (B) EMSA, left panel: the32P-labeled PAI-1 HRE-1/2 oligonucleotide was incubated with either 10 μg protein of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells in the absence or presence of preimmune serum or rat HIF-1 antibody as indicated (cf, Materials and Methods). In EMSA with antibodies, the nuclear extracts were preincubated with 1 μL of the HIF-1 antibody for 2 hours at 4°C before adding the labeled probe. Middle panel: the32P-labeled HRE-2 (-168/-152) oligonucleotide and the PAI-1 HRE-1 (-182/-166) oligonucleotide were incubated with either 10 μg of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells in the absence or presence of preimmune serum or rat HIF-1 antibody as indicated. The inducible (I) HIF-1 complex was seen with the HRE-2, but not with the HRE-1 oligonucleotide. Right panel: the 32P-labeled mutated HRE-2 (-168/-152) oligonucleotide (M-HRE) was incubated with either 10 μg of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells or with extracts from hypoxic cells in the presence of rat HIF-1 antibody as indicated. The image had to be overexposed to visualize the weak constitutive binding. The DNA-protein binding was analyzed by electrophoresis on 5% native polyacrylamide gels. I, induced HIF-1 complex; C, constitutive complex; S, supershifted complex. (C) Western analysis of nuclear extracts from normoxic (16% O2) or hypoxic (8% O2) cells. A total of 20 μg nuclear extract from 2 different preparations was resolved on a 10% SDS gel and probed with preimmune serum or the HIF-1 antibody.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal