Fig 2: EGFR is over-expressed in SCCHN cell lines.qRT-PCR was performed to measure ErbB transcript levels in the three SCCHN cell lines, normalized to that of the normal oral epithelial (NOE) cells. Expression fold change was determined by the 2−ΔΔCt method. ***p<0.001; student's t test.

Fig 3: TUSC3 inhibits EGFR phosphorylationA., Subcellular localization of ectopic TUSC3 protein at the ER. SW480 cells were transfected with FLAG-TUSC3 plasmid, fixed and stained for immunofluorescence microscopy. Color legend: red = FLAG-TUSC3, green = calnexin (ER marker) or phalloidin (actin), blue = nuclei (DAPI). Overlay of TUSC3 with calnexin or actin appears in yellow. Magnification 630x. B., TUSC3 blocks tyrosine phosphorylation of EGFR/Her1. SW480 cells were transfected with TUSC3 and EV plasmids for 24 h, followed by serum removal (“starvation”) for 16 h and a restimulation with 20 % FCS (“serum shock”) for 0 to 3 h before cell harvest. Cells were then subjected to subcellular fractionation (SCF). Western blot analyses were done with an Ab against the C-terminal cytoplasmic (intracellular) domain containing phospho-tyrosine residue Y1068 important for EGFR activity (P-EGFR). O.D. values from bands in gels were calculated as -fold ± S.E. (*p < 0.05 TUSC3 vs. EV, Two-way ANOVA, n = 3). C., TUSC3 inhibits EGFR phosphorylation without affecting total cellular EGFR protein levels. Cells were transfected and treated as in B and total cell lysate (TCL) was subjected to Western blot using the C-terminal Ab against the phosphorylated (Y1068) (*p < 0.05 TUSC3 vs. EV, Two-way ANOVA, n = 3) and unphosphorylated intracellular domain of the EGFR (n.s.). Data are calculated as in B. Similar results were obtained for the transferrin receptor (TFR/CD71). D., TUSC3 knock-down increases EGFR phosphorylation. HCT116 cells were transfected with siRNAs and analyzed for P-EGFR in TCL as in B (*p < 0.05 TUSC3-siRNA vs. control-siRNA, Two-way ANOVA, n = 4). E., Representative Western blots of the experiments in C-D are shown.

Fig 4: Comparing adapter protein performance for split TEV recruitment assays to monitoring ERBB receptor activities. Split TEV recruitment assays for ERBB family receptors. EGFR (a), ERBB2/ERBB3 (c), and ERBB4 (e) activities were assessed in PC12 cells using EGF to stimulate EGFR, and EGF-like domain (EGFld) to stimulate ERBB3 and ERBB4. For split TEV assays, the indicated receptor fusions were transfected together with indicated adapters that were fused to the CTEV moiety. Note that for the ERBB2/ERBB3 assay (c), ERBB2 is co-transfected to allow heterodimerisation and thus ERBB3 phosphorylation, which is required for the recruitment of adapters. Assays were stimulated for 16 h and analysed by a firefly luciferase assay. Non-stimulated samples are shown as open bars and stimulated ones as grey bars. FC fold change, Ctrl control (no adapter transfected). Results are shown as average of six samples, and error bars are shown as SEM. Significance was calculated using the unpaired t test, with **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; n.s. not significant. Precise p values are provided in Table S1. Biochemical validation of the expression of ERBB receptors and adapters. Plasmids encoding EGFR (b), ERBB3 (d), and ERBB4 (f) (all tagged with NTEV-tcs-GV-2HA), ERBB2-V5 (d), and adapter proteins (all tagged with CTEV-2HA) were transiently transfected into PC12 cells, allowed to express for 16 h, and lysed. Lysates were subjected to Western blotting using the indicated antibodies. Calculated sizes of fusion proteins are provided in Table S1. Arrow indicates bands of artificial adapter fusions. Note that SH2(PIK3R1) is only very weakly expressed

Fig 5: Expression level of ErbB family and MET, and activation level of selected receptors in melanoma cell lines(A) qRT-PCR analysis of EGFR, ErbB2, EbrB3, ErbB4 and MET levels. Results are expressed as the mean (relative gene expression compared to GAPDH) ± SD of three independent experiments. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). (B) Log2 expression of selected receptors across 269 melanoma samples (114 primary and 155 metastatic) from GEO database. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). (C) Western blot analysis of pEGFR/EGFR and pMET/MET in cells incubated with culture medium alone or containing EGF, HGF or both for 4 h. Membranes were probed with antibodies against total and phosphorylated EGFR and MET and are representative for at least three independent experiments. GAPDH was used as the sample loading control.