Rbp4 and Fest are expressed shortly after the onset of spermatocyte development and interact physically. (A) Schematic of germ cell development in the Drosophila male. Due to space constraints, only one spermatocyte (red brackets) is shown advancing through the meiotic and post-meiotic stages. Orange, hub; blue, germline stem cell; gray, spermatogonia; dark red, germ cells undergoing premeiotic S DNA replication; turquoise, spermatocytes; yellow, meiotic divisions; green, round spermatids. (B,C) Anti-GFP immunofluorescence of testes from flies expressing (B) Rbp4-eYFP or (C) eYFP-Fest. Scale bars: 100 µm. (D,E) Anti-GFP (green) and anti-HA (red) immunofluorescence on testes from flies expressing (D) Rbp4-eYFP, Bam-HA or (E) eYFP-Fest, Bam-HA. Scale bars: 50 µm. (F) RT-PCR on fest transcript from RNA from males, females and gonadectomized males. Control: GAPDH2. (G) Anti-GFP, anti-HA western blot of anti-GFP immunoprecipitations from testes of Rbp4-HA flies and eYFP-Fest, Rbp4-HA flies. (H) Anti-HA, anti-Myc western blot of anti-HA immunoprecipitations (left panels) or anti-Myc immunoprecipitations (right panels) from S2 cells transfected with either HA-Fest, Myc-Rbp4 or both, as indicated.