Pierce coomassie plus protein assay kit

Buffer [50 mM ammonium bicarbonate, 0.2% w/v RapiGest SF surfactant (Waters; Elstree, United Kingdom)] was added to washed enteroid pellets before sonication using sonicating water-bath (Jencons Scientific; Leighton Buzzard, United Kingdom) for 3 × 10 min on ice. Samples were then heated at 80°C for 10 min. Protein content was determined using the Pierce™ Coomassie plus protein assay kit (ThermoFisher) according the manufacturer's instructions. Protein content was normalized between samples using 50 mM ammonium bicarbonate. Protein were then reduced with 3 mM dithiothreitol (Sigma) at 60°C for 10 min then alkylated with 9 mM iodoacetimde (Sigma) at room temperature for 30 min in the dark. Proteomic grade trypsin (Sigma) was added at a protein: trypsin ratio of 50:1 and samples incubated at 37°C overnight. RapiGest was removed by adding tricholoroacetic acid to a final concentration of 1% (v/v) and incubating at 37°C for 2 h. Peptide samples were centrifuged at 12,000xg for 60 min (4°C) to remove precipitated RapiGest.

Protein lysates were prepared from treated tissue culture cells in CLB1 buffer (Zeptosens/Bayer, Leverkusen, Germany), and protein was quantified using a Pierce™ Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific) (n = 3 per treatment). Details of the RPPA protocol are given in supplementary material, Supplementary materials and methods.