Was Roxadustat Worth The Dough?

We validated the specificity of the antibody using a number of assays (see Extended Experimental Procedures; Figures S4A�CS4C) and showed that it can also detect the secreted form of the protein in 293T cell cultures ( Figure?S4D). Western blot Adenylate cyclase assays of protein extracts from the?brain of Df(16)A+/? mice and WT littermates showed the expected increase (25%) in Mirta22 levels in mutant mice ( Figure?4B). A similar magnitude increase of the Mirta22 immunocytochemical signal was observed in Df(16)A+/? cultured neurons, as compared to WT neurons ( Figure?4C). Immunostaining of neuronal cultures showed that Mirta22 is primarily a neuronal protein (Figure?4D, top). At the subcellular level, it is found primarily Androgen Receptor Antagonist in the soma, where it colocalizes with the Golgi apparatus marker GM130 (Nakamura et?al., 1995). As the neurons mature, it is also found in vesicles and tubular-like clusters within the dendritic shafts (Figure?4D, middle). Mirta22 immunoreactivity was not detected in cultures stained with preimmune serum (Figure?S4E) and was diminished by 64% in Mirta22 shRNA-transfected neurons ( Figures S4F and S4G, lower panel). Immunohistochemical analysis demonstrated that Mirta22 is widely distributed in the brain, where it is localized in neurons ( Figure?4D, bottom). Accumulating evidence suggests that miRNAs may target functionally connected genes, often in a developmental stage-specific manner (Tsang et?al., 2010; Zhang et?al., 2009). Consistent with this notion, functional annotation Roxadustat concentration clustering analysis of?2,695 out of 2,708 predicted miR-185 targets (TargetScan Mouse v.5.2) included in the DAVID Mus musculus gene functional annotation database (http://david.abcc.ncifcrf.gov) identified as the top enriched gene cluster (gene count?= 159, Enrichment Score?= 8.56, FDR-corrected p?= 2?�� 10?9) the Gene Ontology (cellular component) term ��Golgi apparatus�� ( Figure?5A). Gene set enrichment analysis (GSEA) on the 2,708 predicted miR-185 targets ranked based on the gene expression profile of Df(16)A+/? mice also indicated that the Gene Ontology terms ��Golgi apparatus part�� and ��Golgi apparatus�� were among the top 20 gene sets in the adult HPC ( Figure?5A). A global perspective on the enrichment of this miR-185 target gene set among the differentially expressed genes in the Df(16)A+/? mice showed a significant enrichment in the adult HPC expression profile (p?= 5?�� 10?4) where, as expected, most of the top genes were upregulated (n?= 34), and only four genes were downregulated (p?