The role of HnrnpF/H as a driver of Oligoteratozoospermia.
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Data of figures showing origional blots for the paper
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Sperm pellets were resuspended in a lysis buffer consisting of 1% (w/v) C7BzO [3-(4-Heptyl) phenyl-3-hydroxypropyl) dimethylammoniopropanesulfonate], 7 M urea, 2 M thiourea, and 40 mM Tris (pH 10.4) at a final concentration of 20 x 106/ 100 µL and incubated for 1 h (4°C) with constant rotation. Supernatant (18 000 × g, 15 min, 4°C) was recovered and total protein was quantified using a 2-D quant kit (G.E. Healthcare, Sydney, Australia) following manufacturer's protocol. All strains were cultured on molasses-based food at 25⁰C except for GAL4 crosses, which were raised at 29⁰C. UAS-Gal4 (Kyoto Drosophila Stock Centre 108-492) was combined with GAL4::VP16-nanos.UTR (BL4937) to generate UAS-Gal4; nosGal4. Bam-Gal4 (Bam-Gal4:VP16, BL80579), ey-Gal4 (BL5534) and ProtB-GFP (BL58406) were obtained from the Bloomington Drosophila Stock Center. GloGFP (v318719) was obtained from the Vienna Drosophila Resource Center. β-tubulinGFP was obtained from L. Quinn and w1118 from WellGenetics, Inc. Generation of UAS-Glo transgenes The Glo cDNA was amplified from an embryo cDNA library and directionally cloned using NotI and XbaI into pUAST. This vector was used to generate transgenic strains by BestGene, Inc. Three separate insertion strains designated UASGlo.M2, UASGlo.M5 and UASGlo.M6 were used in this study. Immunostaining and microscopy Testes were dissected from adult flies in PBS. They were either visualized live under phase contrast microscopy using a Zeiss Axioskop 2 microscope and images captured with Zeiss Zen software or fixed in 4% formaldehyde diluted in PBT (PBS + 0.2% Triton X-100 (Sigma)) for 20 minutes. Testes were then washed and blocked in PBT + 5% normal horse serum for 45 minutes before being incubated overnight at 4C in primary antibodies (goat anti-Vasa, 1:100, Santa Cruz Biotechnology Cat# sc-26877, RRID:AB_793877), rat anti-DE-cadherin (DSHB, 1:100, Cat# DCAD2, RRID:AB_528120) diluted in PBT, washed in PBS and incubated in secondary antibodies diluted in PBS for 2 hours at room temperature. After washing testes were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen) and imaged using a Zeiss AiryScan LSM800 or LSM880 laser scanning confocal microscope. Drosophila Sperm quantification Control GAL4 and UAS-Glo strains were combined with ProtB-GFP to label sperm heads and seminal vesicles were separated from live testes in PBS. Seminal vesicle contents were spread in a drop of Ringer’s buffer (20uL) on a microscope slide and imaged with a Zeiss Axioskop 2 and Zen software. Sperm numbers were counted using FIJI image analysis software. Non-overlapping GFP-labelled bent sperm heads were manually counted. Immunoblotting: Immunoblotting was performed using 5 ug of protein. Nitocellulose blots were stained with anti-CAMP (Proteintech Cat# 12009-1-AP, RRID:AB_908736), anti-HSP4AL (Abcam Cat# ab87241, RRID:AB_2119828) and anti MENT (Sigma-Aldrich Cat# HPA037430, RRID:AB_2675473).