Kc167 cells were transfected with one of 16 GFP-tagged proteins from the insulin receptor - target of rapamycin pathway. Cells were grown in medium containg 10% FBS, then starved (2% FBS) and treated with copper overnight (to induce transfected tagged bait proteins). Cells were either treated with 100nM insulin for 20 minutes before harvest or left untreated. All conditions were done in biological duplicate.
Cells were lysed in the presence of DSP cross-linker and purified using anti-GFP beads.
Purified proteins were characterized by mass spectrometry.