Figure 3.
Analysis of the YB-1 interactions with VNTR domains in EMSA. A, Bacterially expressed YB-1 can form specific complexes with the 32P-labeled Stin 2.9, Stin 2.10, and Stin 2.12 DNA. Each incubation mixture contained the same amount of nonspecific competitor poly (deoxyinosinic-deoxycytidylic) acid (dIdC) (0.1 μg); 1 μg of GST-YB-1 fusion was added to samples in lanes 1, 6, and 11; 2 μg of GST-YB-1 was added to samples in lanes 2, 7, and 12; 4 μg of GST-YB-1 was added to samples in lanes 3-5, 8-10, and 13-15. The specific competitor mdr-1 oligonucleotide was added as follows: lanes 4 and 9, and 14- to 50-fold molar excess; lanes 5 and 10, and 15- to 100-fold molar excess. The positions of DNA-protein complexes are indicated (labeled 1, 2, and 3). B, Competition EMSA analysis. The labeled Stin 2.12 DNA fragment was incubated with the same amount of nonspecific competitor dIdC (0.1 μg) and 2 μg of GST-YB-1 protein in all reactions (except 6, in which a 2 μg of GST component of the fusion protein was added). Competitors tested in this experiment were as follows: lane 2, 100-fold molar excess of rp 6/7 oligonucleotide; lane 3, 100-fold molar excess of rp 3/4 oligonucleotide; lane 4, 0.5 μg of baculoCTCF; lane 5, 1 μg of baculoCTCF; lane 6, 2 μg of GST; lane 7, 100-fold excess of the mdr-1 mutated oligonucleotide that is unable to bind YB-1. C, EMSA analysis of the competition with rp 3/4-GST-YB-1 complexes. Incubation mixtures contained labeled rp 3/4 oligonucleotide, nonspecific competitor dIdC (0.1 μg), and 2 μg of GST-YB-1. DNA-protein complexes were preformed during a 30 min incubation on ice, then the competitors were added, and incubation continued for an additional 20 min. Competitors were added as follows: lane 1, no competitor; lanes 2-5, 10-, 25-, 50-, and 100-fold molar excess of oligonucleotide mdr-1, respectively; lanes 6-9, 0.25, 0.5, 1, and 2 μg of baculoCTCF, respectively. D, EMSA analysis of the competition with rp 6/7-GST-YB-1 complexes. The incubation mixtures contained labeled rp 6/7 oligonucleotide, nonspecific competitor dIdC (0.1 μg), and 2 μg of GST-YB-1. In C and D, DNA-protein complexes were preformed during a 30 min incubation on ice, then the competitors were added, and incubation continued for an additional 20 min. Competitors were added as follows: lane 1, no competitor; lanes 2-8, 10-, 25-, 50-, 100-, 150-, 200-, and 250-fold molar excess of oligonucleotide mdr-1, respectively; lanes 9-12, 0.25, 0.5, 1, and 2 μg of baculoCTCF, respectively.