Complete without edta

Worms were grown on either control or smk-1 RNAi bacteria until day 2 of adulthood and then harvested into a lysis buffer (20 mM MOPS (pH 7.2), 15 mM MgCl₂, 100 mM NaCl, 1% (v/v) Triton X-100, 1 mM DTT, Complete without EDTA (Roche), 1 mM phenylmethyl sulphonyl fluoride, and PhosSTOP (Roche)). Samples were lysed using a FastPrep and silicon carbide beads with 1.0 mm diameter (BioSpec). Per condition, material equivalent to 50 worms was loaded onto SuperSep Phos-tag (50 µM) 10% precast gels (Wako) and then analyzed by SDS-PAGE and western blotting. Monoclonal mouse anti-GFP antibody (Roche) diluted 1:1000 in TBST containing 5% milk powder was used to detect DAF-16::GFP in the different samples.

The cDNA of pph-4.1 was cloned into the GST-fusion expression vector pGEX-4T1 and the resulting plasmid transformed into BL21(DE3)-RIPL (Agilent). The resulting bacterial strain was grown to an OD₆₀₀ of 0.6 at 37 °C and 230 rpm and then induced with IPTG for 18 h at 18 °C and 230 rpm. Next the bacteria were harvested and frozen in liquid nitrogen. For the purification of PPH-4.1, the bacteria were thawed into TBS buffer containing 1 mM EDTA, 1 mM DTT, and Complete without EDTA (Roche), and incubated with lysozyme for 30 min at 4 °C. Resulting lysate was sonicated, Triton X-100, RNase A (Roche), and DNase I (Invitrogen) were added to concentrations of 1% (v/v), 10 µg ml⁻¹, and 5 µg ml⁻¹, respectively, and the lysate then incubated for another 30 min at 4 °C. Glutathione-Sepharose 4B resin (GE Healthcare) was added to the lysate and incubated rotating for 2 h at 4 °C. Finally, the resin was washed in TBS, resuspended in TBS, and then used for in vitro phosphatase assays.

Growth of C. elegans to large scale, harvesting, lysis, IP of the bait proteins, and eventual analysis of the precipitated material by tandem mass spectrometry were conducted as previously described^{17 (link)}. At first, the animals were grown asynchronously at 15 °C to scale, then shifted for 16 h to 25 °C and harvested. Animals were transferred to lysis buffer (50 mM HEPES at pH 7.4, 1 mM EGTA, 1 mM MgCl2, 150 mM KCl, 10% (v/v) glycerol, Complete without EDTA (Roche), 1 mM phenylmethyl sulphonyl fluoride, and phosphatase inhibitors (Sigma)) and lysed by grinding under liquid nitrogen. NP-40 was added to 0.05% (v/v), and the resulting lysate was cleared at 20,000 g. mCherry-tagged bait proteins were immunoprecipitated using a rabbit-anti-mCherry antibody that we generated in the lab and coupled to Protein A resin (Biorad). GFP-tagged bait proteins were immunoprecipitated using GFP-trap resin (Chromotek). Immunoprecipitated proteins were eluted using 100 mM glycine at pH 2.6. As negative controls for background subtraction, the same purifications and later mass spectrometry analyses were conducted using wild type (untagged) C. elegans.