Ezview anti flag m2

Cells were harvested (500 x g, 3 min), lysed in lysis buffer (150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 5 mM EDTA, 50 mM Tris pH 7.5), sonicated for 3–5 pulses at 10% intensity, and centrifuged for 10 min at 13000 x g. A fraction of the supernatant was saved, quantified using the BCA assay (Thermo Fisher), and normalized as input, and the remainder was immunoprecipitated by rotation at 4°C overnight using either anti-GFP-nanobody Sepharose (Chromotek), EZview anti-FLAG-M2, or EZview anti-HA resins (Sigma). For immunoprecipitation using the soluble DVL3 antibody, the sample was incubated with primary antibody for 1 h at 4°C with rotation, followed by rotation overnight at 4°C with Protein G Sepharose (BioVision). The resin was then centrifuged for 10 min at 1000 x g, washed three times with lysis buffer and analyzed by SDS-PAGE and western blot, with detection by chemiluminescence (using SuperSignal West Pico (Thermo) or Clarity (Bio-Rad)) or, as described below in detail, mass spectrometry-based proteomics.