Western blot analysis

Animals were grown synchronously to day 1 adults and collected in M9 buffer. After three washes, pellets were snap-frozen in liquid nitrogen and stored at −80°C. To lyse the worms, pellets were put in radioimmunoprecipitation assay buffer, ground twice using a TissueLyser at 75 Hz for 6 min at low temperature, and then centrifuged at 10,000g at 4°C to collect the supernatant. Protein concentrations were measured using the BCA Protein Assay Kit. Proteins were separated using SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membranes were blocked in 5% milk and then incubated with primary antibodies to H3K9me3 (1:1000; Abcam, ab176916), H3 antibody (1:10,000; Cell Signaling Technology, H9715), GFP (1:5000; Roche, 11814460001), or actin (1:5000; Sigma-Aldrich, A1978). Then, protein was visualized using horseradish peroxidase–conjugated anti-rabbit (1:5000) or anti-mouse (1:5000) secondary antibody and ECL Western Blotting Substrate.