Nucleus-specific protein function for AGO2 in male germ cells. (A) Western blot for AGO2 protein in whole-cell lysate (WCL), cytoplasmic fractions, or nuclear fractions of meiotic (pachytene spermatocyte, SYCP3+) and postmeiotic (round spermatid, ACRV1+) male germ cells. Tubulin and histone H3 are found in cytoplasmic and nuclear fractions, respectively. Round spermatids have not yet begun histone-to-protamine exchange and therefore have similar levels of histone H3 compared with meiotic cells. (B) Immunoprecipitation of AGO2 for IP-MS in cytoplasmic and nuclear germ cell fractions validated by western blotting with anti-AGO2 antibody. One percent of total lysate was used as input. IP with mouse IgG is a negative control. (C) Common and compartment-specific AGO2 protein interactors in the nucleus and cytoplasm of wild-type postmeiotic cells. Box shows detected miRNA/siRNA pathway components along with the number of unique peptides identified in each compartment. (D) Statistically enriched Gene Ontology (GO) terms for AGO2 protein interactors in the nuclear compartment.
