GFP IP

IPs with GFP-tagged proteins were performed with GFP-binding magnetic agarose beads (GFPtrap MA, Chromotek, #gtma-20). Per IP sample, 10 µl of bead slurry was used and washed two times with 500 µl Wash Buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1:1000 Pepstatin A/Leupeptin, 1:100 PMSF). Afterwards, the beads were resuspended in 450 µl Wash Buffer and up to 1 mg of complete extract of the respective C. elegans strain (of mixed-stage embryos or young adults) was added to a final volume between 500 and 750 µl. The IP samples were incubated at 4 °C rotating for 2 h. Following three washing steps with 500 µl Wash Buffer the beads were resuspended in 1x LDS (4x NuPAGE LDS sample buffer, Thermo Scientific, #NP0008) supplemented with 100 mM DTT and boiled at 70 °C for 10 min. When used for mass spectrometry, the samples were prepared in quadruplicates per strain/condition. In the IP-MS related to Supplementary Fig. ^{5e, f}, the Wash Buffer was supplemented with 2 mM MgCl₂ and 0.05% of recombinant endonuclease from Serratia marcescens, or Sm nuclease⁷³, produced by the IMB’s Protein-Production Core Facility.