A02020

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.

Proteins were separated by SDS-PAGE. Gels were blotted onto a PVDF membrane (Merck Millipore, Burlington, MA, USA) with transfer buffer at 64V for 2h. Membranes were blocked for 1 h at room temperature, followed by washing. The antibodies—anti-GFP (1:2,000; #A02020; Abbkine, Wuhan, China), anti-RFP (1:2,000; #A02120; Abbkine), anti-His (1:3,000; #A02050; Abbkine), or anti-GST (1:2,000; #A02030; Abbkine)—were added and incubated at 4°C overnight, followed by three washes. Membranes were then incubated with goat anti-mouse antibody (ab6789; Abcam), or goat anti-rabbit (ab205718; Abcam) at a ratio of 1:10,000 in the blotting buffer at room temperature for 2 h. After three washes, membranes were incubated with chemiluminescence HRP substrate (#WBKLS0100, Merck Millipore) for 5 min, and then visualized by excitation at 780 or 800 nm.