T5168

Stage-synchronized animals for control and experiment groups were picked (N > 40) and lysed directly into 20 μl of Laemmli sample buffer for Western blot analysis. Proteins were resolved by 15% SDS–polyacrylamide gel electrophoresis (PAGE) (Bio-Rad, 4561084) and transferred to a nitrocellulose membrane (Bio-Rad, 1620167). Proteins of interest were detected using antibodies against GFP (A02020, Abbkine), tubulin (Sigma-Aldrich, T5168), and H3 (Abcam, ab1791). All experiments were repeated for multiple times.
For subcellular fractionation, 50-ml adult-stage animal pellets were washed with M9 buffer three times and resuspended in 500 μl of RIPA lysis buffer (Amresco, N653) with 10 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (BioTools, B14002). Then, pellet samples were disrupted by TissueRuptor (motor unit “8” for 1 min) and incubated for 45 min in a 4°C cold room. The lysate was centrifuged at 13,000 rpm for 15 min, the supernatant was collected as the soluble part, and the pellet was resuspended in 500 μl of RIPA lysis buffer with 10 mM PMSF and protease inhibitor cocktail as the insoluble part. Samples (20 μl) added with equal volume of 2× Laemmli sample buffer were subject to Western blot analysis, as described above.