The lack of NF-YA subunit in cell extracts from freshly isolated monocytes is not due to degradation. Western blot analysis (30 μg) from freshly isolated monocytes (d0) and fully differentiated macrophages (d7) (lanes 1 and 2) was performed with an affinity-purified antibody against NF-YA. Extracts were prepared, as indicated in Materials and Methods, but without the addition of protease inhibitors. In lanes 3 and 4, increasing amounts of whole cell extract from freshly isolated monocytes (d0) were added to 30 μg of cell extracts from d7 cultured monocytes, incubated for 40 minutes at room temperature, and then subjected to Western blot analysis.