(A) Western blot analysis of E14.5 male gonads from embryos with Nanos2+/−, Nanos2+/− with the transgene, Nanos2−/−, and Nanos2−/− with the transgene using antibodies against NANOS2 (upper panel), NANOS3 (2nd panel), DAZL (3rd panel), and TUBULIN (lower panel). DAZL is a loading control for germ cells, whereas TUBULIN is a loading control for the total amount of protein. Note that DAZL is up-regulated in Nanos2−/− male gonads as described previously (Suzuki et al., 2010). (B) Western blot analysis of male gonads from embryos with Nanos2+/−, Nanos2−/−, and Nanos2−/− with Flag-tagged NANOS2-ZM at E13.5, E14.5, and E15.5 using antibodies against NANOS2 (upper panel), NANOS3 (middle panel), and TUBULIN (lower panel). (C–E) Sections of male gonads from Nanos2−/− embryos at E15.5 were immunostained with antibodies against NANOS3 (green) and DCP1a (red). DNA was counterstained with DAPI (blue). Arrowheads indicate the localization of NANOS3 in P-bodies. Scale bar, 50 µm in C for C–E.