Afatinib

The water-soluble tetrazolium (WST-1) proliferation assay (Roche Diagnostics) was used to measure cell proliferation after knockdown or stimulation as described earlier [17 (link)]. Cells were treated with cetuximab (1/10 µg/ml, Merck), trastuzumab (5/20 µg/ml, Roche), afatinib (0.5 µM, Biozol), DMSO (0.05%, afatinib solvent), trastuzumab solvent (described in [17 (link)]) or cetuximab solvent (8.48 mg/ml NaCl, 1.88 mg/ml Na₂HPO₄ × 7H₂O, 0.41 mg/ml NaH₂PO₄xH₂O) for 72 h (cell numbers see Table S1, Additional file 1). In case of stimulation, cells were treated with 5 ng/ml recombinant HBEGF or 15 ng/ml recombinant AREG (R&D Systems).

Western blot analyses were performed according to a standard protocol described earlier [16 (link), 17 (link)]. One day after transfection, cells were harvested and plated. The following day they were treated for 20 minutes with 0.5 μM afatinib (Biozol) or 0.05% DMSO (solvent control). 15–25 μg of protein was loaded for each lane. Antibodies against MET D1C2 (#8198, 1:1000 in 5% BSA), pMET Y1234/1235 (#3077, 1:1000 in 5% BSA), EGFR (#2232, 1:1000 in 5% skimmed milk), pMAPK T202/Y204 (#9101, 1:2000 in 5% skimmed milk), pAKT S473 (#4060, 1:2000 in 5% BSA), anti-rabbit IgG (#7074, 1:2000 in TBST) (all Cell Signaling Technology), pEGFR Y1068 (#44788G, 1:2000 in 5% skimmed milk, Thermo Fisher Scientific), β-Actin (#A1978, 1:5000 in 5% BSA, Sigma-Aldrich) and anti-mouse IgG (#NA931, 1:10000 in 5% skimmed milk, GE Healthcare, distributed by VWR) were used. The proteins pMET and pMAPK as well as EGFR and pAKT were analyzed on the same membrane.

Western blot analyses were performed as described earlier [40 (link), 41 (link)]. MKN1, MKN7, Hs746T and NCI-N87 cells were treated for 24 h with EGF (5 ng/ml, Sigma Aldrich), cetuximab (1 μg/ml, Merck), trastuzumab (5 μg/ml, Roche) or afatinib (0.5 μM, Biozol). Antibodies against pAKT (1:2000 in 5% BSA TBST,# 9271, Cell Signaling Technology), pMEK1/2 (1:1000 in 5% BSA TBST, # 9154, Cell Signaling Technology), ppMAPK3 (1:2000 in 5% milk TBS-T,# 9101, Cell Signaling Technology), pp70S6K (1:1000 in 5% BSA TBST,# 9205, Cell Signaling Technology), cleaved caspase 3 (Asp 175) (1:1000 in 5% skimmed milk TBST, #9664, Cell Signaling Technology), α-tubulin (1:10000 in 5% skimmed milk TBST, #T9026, Sigma-Aldrich), anti-rabbit IgG (1:2000 in TBST, #7047, Cell Signaling Technologies) and anti-mouse IgG (1:10000 in 5% skimmed milk TBST, #NA931, GE healthcare, distributed by VWR) were used.