antibody to PU.1 in alcohol-fed rats only (panel B). Next, we extended these findings to alveolar macrophages that were freshly isolated from alcohol-fed rats whose diets were supplemented with zinc in vivo and looked for a comparable supershift in the Nrf2 band when the anti-PU.1 antibody was added. The supershift assay in Figure 7 is qualitatively similar to panel A in Figure 6, providing evidence that dietary zinc treatment promotes cooperative binding of PU.1 and Nrf2 in the alveolar macrophages of alcohol-fed rats in vivo and therefore that the findings in Figure 6 are not restricted to zinc treatment of cells in vitro. Interestingly, as shown in Figure 7 there did not appear to be any significant interactive binding by Nrf2 and PU.1 in the alveolar macrophages of control-fed rats, suggesting that under normal conditions the ARE and GM-CSF signaling pathways may be less interdependent than during stresses such as chronic alcohol ingestion.
