Electrophoretic mobility shift assay (EMSA) for PU.1 and Nrf2 nuclear binding was performed to determine nuclear binding in alveolar epithelial cells and macrophages. This procedure has been previously described. (Joshi et al., 2006;Joshi et al., 2005) In brief, macrophages were first isolated as described above, and nuclear protein extracts were prepared. Double-stranded PU.1 consensus oligonucleotide (5′ TGA AAG AGG AAC TTG GT) or Nrf2 consensus oligonucleotide (5′ TGG GGA ACC TGT GCT GAG TCA CTG GAG) was radiolabeled with 32P gamma ATP using T4 polynucleotide kinase enzyme. Nuclear protein (5 ug) was incubated with radiolabeled PU.1 or Nrf2 for 30 min at room temperature. When applicable, 2 μg of an anti-PU.1 or an anti-Nrf2 antibody was added to determine if there was a supershift assay (reflective of PU.1 and Nrf2 binding to the same oligonucleotide). After binding, protein-DNA complexes were electrophoresed on a native 4.5% polyacrylamide gel using 1x Tris-glycine buffer, and dried under vacuum prior to x-ray film exposure.
