Chemiluminescence luminol

Proteins were extracted by homogenizing animals in RIPA buffer supplemented with 10% protease inhibitor cocktail (Sigma‐Aldrich). The homogenates were cleared by centrifugation, and the supernatants were subjected to electrophoresis through 10% sodium dodecyl sulfate/polyacrylamide (SDS/PAGE) gels containing pre‐stained protein markers and transferred to polyvinylidene fluoride filters (Bio‐Rad Laboratories, Hercules, CA, USA) (Harlow & Lane 1988). The filters were incubated with 5% non‐fat dry milk in TBST buffer (50 mmol/L Tris/HCl, pH 7.6, 100 mmol/L NaCl, 0.1% Tween‐20) to block non‐specific binding, washed three times for 5 min in TBST, incubated overnight in a 1:100 dilution of PIWI polyclonal antibody (ab5207, Abcam, Cambridge, MA, USA) in TBST at 4°C, and finally incubated for 1 h at room temperature in a 1:25000 dilution of horseradish‐peroxidase‐conjugated anti‐rabbit IgG secondary antibody. After three washes for 5 min with TBST, the signals were visualized with Chemiluminescence Luminol (Santa Cruz Biotechnology, Santa Cruz, CA, USA) used according to the supplier's specifications. Images of X‐ray films were taken with a digital camera and scanned, and band density was calculated by comparison with a glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gel loading standard (Sigma‐Aldrich).