EMSA with ISRE and pIRE oligonucleotides using nuclear extracts obtained from NB4 and HeLa cells treated with ATRA and IFNα. (A) Nuclear extracts obtained from NB4 or HeLa cells treated for various lengths of time with the indicated stimuli were incubated with (32P)-radiolabeled ISRE, pIRE, or Sp1 double-stranded oligonucleotides in the absence (−) or in the presence (+) of a 250-fold excess of the respective cold oligonucleotide (Sp. comp.) or the same concentration of a negative-control oligonucleotide containing mutated GAS and ISRE consensus sequences (GAS/ISRE mutant oligonucleotide, Santa Cruz) (non-Sp. comp.) and subjected to EMSA. Specific retarded complexes are indicated with arrows on the left. An EMSA representative of three independent experiments is shown. (B) Nuclear extracts obtained from NB4 cells treated for 4 days with medium alone (Medium) and medium containing ATRA or IFNα were preincubated for 30 minute at 4°C either in the absence (−) or in the presence of the indicated antibodies. Following this preincubation, the extracts were incubated with (32P)-radiolabeled ISRE and pIRE for a further 20 minute at room temperature, and subsequently subjected to EMSA. Specific and nonsupershifted retarded bands are indicated by arrows. An EMSA representative of two independent experiments is shown. In all the experiments, ATRA was used at a concentration of 10−6 mol/L and IFNα at a concentration of 1,000 U/mL.
