Upon 2 unique peptide matches to prevent false

Upon EGF stimulation, EGFR in Her14 cells will be activated by the
formation of dimers, tetramers, or higher-order oligomers. This could either
induce several downstream signal transduction pathways or it may induce
endocytosis. According to Seedham et al, higher-order oligomerization starts by
the formation of dimers by a back-to-back dimerization interface located on
domain II, followed by repeated incorporation of additional dimers using the
face-to-face interface at EGFR ectodomain I and III. The clustering of EGFR
oligomers leads to a negative membrane curvature organized by the dimer
transmembrane helices, which will function as hydrophobic wedges in the plasma
membrane. This EGFR clustering-induced membrane invagination will recruit
curvature sensitive proteins, such as F-BAR domain-containing FCHO
proteins.  By isolating these activated
EGFR oligomer clusters using SMA copolymer, we are able to determine the relative
abundance of proteins recruited in the first steps of endocytosis. As
previously mentioned, the EGF stimulated EGFR-enriched nanodiscs showed
enhanced Tyr-1068 phosphorylation indicating trans-phosphorylation of kinase
domains (figure 3A). Because EGFR was used as bait during the
immunoprecipitation, the NSAF of each protein was normalized by the NSAF of
EGFR (see methods for further details). The relative protein abundance in the
stimulated EGFR interactome was based on the ratio between the EGF-induced NSAF
of proteins against the control NSAF of proteins (supplementary table 2 and 3).

Table 1 provides an overview for a total of 72 proteins with an enrichment
factor of ³ 1.5 containing at least 2 unique peptide
matches to prevent false positives in the EGF stimulated sample. Here, MYB-binding protein
P160 1a (Mybbp1a) shows the highest enrichment
value of 4.375. The enrichment value could not be calculated for twenty
proteins due to the absence of unique peptide matches in the control sample.

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Nonetheless, they are taken into account based on the presence of ³2 unique peptide matches in the EGF-stimulated sample.