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.

Following protein identification, spectral counts for proteins were normalized and the SAINT algorithm was used to assign a confidence score. High confidence interactions were determined as follows. First, interactions with a SAINT score greater than 0.99 were selected. Then, additional interactions between the 58 factors identified in the first step, where SAINT score was at least 0.80, were added. Interactions involving 17 common ribosomal, cytoskeletal or heat shock protein contaminants were filtered out.