Alcohol has independent effects on the GM-CSF/PU.1 pathway and the ARE/Nrf2 pathway. Our previous studies have always examined these pathways as separate entities, and it is not currently known whether there is any interaction or crosstalk between these two important systems. Therefore, we explored the possibility of cooperative interaction by performing electrophoretic mobility shift assays (EMSA), but with the addition of antibodies to Nrf2 and PU.1 to look for a supershift (see Methods). First, we examined the effect of zinc treatment in vitro on alveolar macrophages. As shown in the representative gel in Figure 6, zinc treatment appeared to increase interactive binding of DNA by PU.1 and Nrf2, as indicated by a supershift of the PU.1 band in the presence of the antibody to Nrf2 (in panel A) and the supershift of the Nrf2 band in the presence of the 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
