We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 μm holes of the support films. Randomly biotinylated 70 S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotinbinding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which wellordered, single crystals are observed to span over entire, 2 µm holes of the support films. Randomly biotinylated 70 S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

FOXO transcription factors have a highly conserved role in regulating transcription of genes involved in differentiation, cell cycle arrest, apoptosis and DNA repair. Loss of FOXO3 in mice has previously been shown to result in a myeloproliferative disease. In agreement with this, we found that an independent Foxo3 null mouse strain, Foxo3 Kca, exhibits an increase in neutrophils in the spleen, bone marrow and blood. This coincides with an expansion of myeloid progenitor cells including pre-granulocyte-macrophage progenitors (Pre-GMs) and granulocyte-macrophage progenitors (GMPs). Surprisingly, despite neutrophilia, the severity of passive serum transfer arthritis was markedly attenuated in Foxo3 Kca mice. These defects appear to be at least partially intrinsic to the myeloid lineage, as deleting Foxo3 specifically from myeloid cells using LysMCre also leads to an elevated number of neutrophils and protection from K/BxN-serum transfer-induced arthritis.

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