Anti myc

Protein samples for western blotting were diluted with 2× sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 19% glycerol, 200 mM DTT, and 0.01% bromophenol blue]. The samples were separated by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and then blotted onto Nitrocellulose Membrane (FUJIFILM Wako Pure Chemical) or Immobilon-P membrane (Merck). After blocking with 5% skim milk, membranes were incubated with primary antibodies in PBS containing 0.1% Tween (T-PBS) at room temperature. Anti-Mael (this study), anti-Siwi, anti-Spn-E, anti-Qin, anti-Vasa, anti-Ago3 (Nishida et al., 2015 (link)), anti-Vret (Nishida et al., 2020 (link)), anti-Me31B (Nakamura et al., 2001 (link)), anti-Flag M2 (Sigma-Aldrich), anti-β-Tubulin, and anti-Myc (Developmental Studies Hybridoma Bank) antibodies were used as primary antibodies. After washing with T-PBS, membranes were incubated with secondary antibodies in T-PBS at room temperature. Anti-mouse IgG (H+L) HRP (Cappel), anti-mouse Ig HRP TrueBlot ULTRA (Rockland), and anti-rabbit IgG HRP-linked (Cell Signaling Technology) antibodies were used as secondary antibodies. After washing with T-PBS, the membranes were then incubated with ECL Prime Western Blotting Detection Reagent (Cytiva). Images were collected using the ChemiDoc XRS Plus System (Bio-Rad).