cDNAs for 15 components of the RTK-Ras-ERK pathway were cloned into into pMK33-CTAP and stable S2R+ cell lines were generated. Cells were induced overnight with 140uM copper. At least two biological replicates were performed for each bait and condition.

Cells were lysed as described in Veraksa et al., 2005 (FBrf0183909) and in-solution tandem affinity purification was performed as described in Siomi and Siomi, 2005 (FBrf0188010).

Purified proteins were reduced with DTT, alkylated with iodoacetamide, trypsin digested, cleaned of buffer and debris by 'ZipTip' and dried to concentrate peptides. Peptides were analysed by LC-MS/MS. MS/MS spectra were matched to peptides using SEQUEST and a composite database of all translated D. melanogaster open reading frames in FlyBase (r5.4).

From 15 baits, 5009 unfiltered interactions among 1188 proteins were identified. SAINT analysis was used to assign confidence scores (PMID: 21131968). 'Tag-only' purifications were used as negative controls. Using a SAINT cutoff of 0.83 and a false discovery rate of 7.2%, a high confidence, filtered network of 386 interactions among 249 proteins was generated.