Co-immunoprecipitation and western blotting

Synchronized young gravid adult worms were washed 3 times with M9, and embryos were obtained by hypochlorite treatment. Embryos were resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 500 mM NaCl, 0.25% deoxycholate, 10% glycerol, 1% NP-40, 2 mM DTT, 1× EDTA-free protease inhibitor cocktail (Roche, Switzerland), 1xPhosSTOP (Roche)) and frozen in liquid nitrogen. Samples were sonicated with a Bioruptor Pico (Diagenode, Belgium —15 cycles, 30-s sonication, 30-s rest, snap freezing every 5 cycles) and spun down (30 min, max speed) to pellet debris. The supernatant was collected, and prewashed Pierce Anti-HA Magnetic Beads (Thermo Fisher Scientific, USA) were added. Following 2 h or overnight incubation on a rotator at 4°C, the beads were collected using a magnetic stand and washed according to manufacturer’s instructions. HA-tagged proteins were eluted by boiling the beads for 5 to 10 min in sample buffer. Protein concentrations in input samples were measured with the Bio-Rad Protein Assay (Bio-Rad, 5000006), and equal amounts were loaded on SDS-page gels. Western blotting was performed according to standard procedures using 4% to 20% gradient or 12% gels and the LI-COR Odyssey system or ECL for detection. Primary antibodies (anti-HA, Sigma-Aldrich mAb 3F10, 1:1,000; anti-OLLAS, Novus Biologicals NBP1-06713B, 1:1,000) were incubated overnight at 4°C, and IRDye or HRP-conjugated secondary antibodies appropriate for each primary antibody were incubated for 45 to 60 min at RT.