Secondary hrp antibodies

C. elegans were synchronized at 20 °C by bleaching gravid adult animals and maintaining starved L1 larvae for at least 24 h before plating on OP50. For sample collection, animals were harvested after 2 h (L1), 12 h (L2), 32 h (L3), 50 h (L4), and 72 h (gravid adult) on OP50. Number of animals loaded per lane was normalized for actin—~1000 L1s, 800 L2s, 600 L3s, 400 L4s, and 200 gravid adults. Proteins were resolved on 4–12% Bis-Tris polyacrylamide gels (Thermo Fisher), transferred to nitrocellulose membranes (Thermo Fisher), and probed with rat anti-HA-peroxidase 1:1000 (Roche 12013819001), mouse anti-FLAG 1:1000 (Sigma, F1804), mouse anti-actin 1:10,000 (Abcam ab3280), or rabbit anti-CSR-1 1:2000 antibodies^{8 (link)}. Secondary HRP antibodies were purchased from Thermo Fisher. Unedited western blots provided in the Source Data File.

For Western blots, proteins were resolved on 4–12% Bis-Tris polyacrylamide gels (ThermoFisher), transferred to nitrocellulose membranes, and probed with anti-FLAG 1:1,000 [M2 clone] (Sigma-Aldrich F1804), anti-actin 1:10,000 (Abcam ab3280), or anti-Myc 1:1,000 [9E10 clone] (ThermoFisher 13–2500). Secondary HRP antibodies were purchased from ThermoFisher.

The RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract the total proteins, and its quality was measured by BCA kit (Beyotime, Shanghai, China). The proteins were separated by 10% SDS-PAGE, and the targeted proteins were transferred onto the PVDF membranes according to the proteins’ molecular weight. Membranes were then blocked with 5% non-fat milk and were incubated with the primary antibodies against LC3B (1:2000, Santa Cruz, United States), p62 (1:2000, Santa Cruz, United States), GAPDH (1:2000, Takara, Japan), ANXA6 (1:2000, Santa Cruz, United States), TSG101 (1:1500, Takara, Japan), Alix (1:1500, Takara, Japan), and HSP70 (1:1000, Santa Cruz, United States) at 4°C overnight. Then, the PVDF membranes were cultivated with the secondary HRP antibodies (ThermoFisher Scientific, United States) for 2 h at 37°C, and the ECL system was used to visualize the protein bands, which were quantified and analyzed by using the Image J software.