Immunoblotting and Immunoprecipitation of Fly Piwi Proteins

Kc167 cell pellets and pestle-ground fly tissues were lysed by sonication in RSB-200 buffer containing 20 mM Tris–HCl pH 7.4, 200 mM NaCl, 2.5 mM MgCl₂, 0.5% NP-40, 0.1% Triton X-100, and complete EDTA-free protease inhibitors (Roche). Lysates were precleared with centrifugation at 16,000g for 20 min at 4°C. Samples were mixed with SDS-loading buffer and denatured at 70°C for 12 min. Proteins were separated by 4%–12% NuPAGE Bis–Tris and blotted to nitrocellulose membranes (Invitrogen). The following antibodies were used in this study: Aub-83 (Vourekas et al. 2016 (link)), SYM11 anti-sDMA (Millipore), E7 anti-β-Tubulin (Developmental Studies Hybridoma Bank). 4D10 anti-Aub, anti-AGO3, anti-Armi, and anti-Tudor antibodies were a gift from Dr. Mikiko Siomi. Piwi-N7 and Vasa-2 antibodies were produced by immunizing rabbits with the following synthetic peptides: Piwi (MADDQGRGRRRPLNED); Vasa-2 (KREFYIPPEPSNDA). Both were conjugated to KLH protein. Sera were affinity purified with columns containing the immobilized peptides (Genscript). Piwi-N7 successfully detected and immunoprecipitated Piwi protein and associated piRNAs in ovary, embryo, Kc167, and OSC lysates (Fig. 1A–C; Supplemental Fig. S3A–E). Similarly, Vasa-2 antibody detected and immunoprecipitated Vasa protein in ovary lysate (Supplemental Fig. 3F).