Precast protein gel

Animals at the same stage from the control and experiment groups were picked (N>30) into 20 μL Laemmli Sample Buffer with 10% β-mercaptoethanol and lysed directly for Western blot analysis. Protein samples were run with 15% Precast Protein Gel (Bio-Rad, 4561084), except that COL-101::GFP samples were run with 7.5% Precast Protein Gel (Bio-Rad, 4561023), and then transferred to the nitrocellulose membrane (Bio-Rad, 1620167). The membranes were blotted by antibodies against GFP (A02020, Abbkine), mCherry (Invitrogen, M11217), Tubulin (Sigma, T5168) and H3 (Abcam, ab1791).
For subcellular fractionation, three plates (6cm dish) of adult-stage animal pellets were washed with M9 buffer three times for worm samples, were resuspended in 500 μL of RIPA lysis buffer (Amresco, N653) with 10 mM phenylmethylsulfonylfluoride (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 12,000 rpm for 20 min, the supernatant was collected as the supernatant fraction, and the pellet was resuspended in 500 μL of RIPA lysis buffer with 10 mM PMSF and protease inhibitor cocktail as the precipitation fraction. Then, 20 μL Samples added with 4× Laemmli sample buffer were subject to Western blot analysis, as described above.

For protein complex immunoprecipitation (co-IP), cells were seeded in 100-mm² dishes (1 × 10⁶/well). After 36 h, transfections were performed using Lipofectamine 2000 (Invitrogen; 11668) according to the manufacturer’s protocol. The transfected cells were allowed to grow for another 36 h before the cells were lysed with 600 µl of immunoprecipitation (IP) lysis buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA; 87787) and centrifuged at 13,000×g for 10 min. The supernatant was incubated with 50 µl of anti-FLAG M2 affinity gel (Sigma; A2220) or protein G-agarose (Roche, IN, USA; 11719416001) for 4 h. The beads were washed five times with 1 × Tris-buffered saline buffer. The proteins were eluted with Laemmli sample buffer (Bio-Rad; #161-0747) and boiled at 95 °C for 20 min. A quarter of the total eluate was resolved using SDS-PAGE with precast protein gels (Bio-Rad; #456-1094).