Edge plates

Cells were seeded in 96-well plates (Edge-plates, Thermo Fisher Scientific, Waltham, MA), serum-starved for 6 h by replacing the medium with DMEM containing 0.1% bovine serum albumin (BSA), and stimulated with EGFR ligands (100 nM AREG, 10 nM BTC, 100 ng/mL EGF, 250 ng/mL EPGN, 10 nM EREG, 10 nM HB-EGF, or 50 ng/mL TGFα). One hour prior to stimulation, the cells were treated with the following compounds at the indicated concentrations (see also Suppl. Table S1): Akt inhibitor VIII (200 nM), 18 PD153035 (100 nM), 19 CAS 879127-07-8 (10 µM), 20 Et-18-OCH3 (5 µg/mL), 21 JNK inhibitor II (1 µM), 22 LY294002 (10 µM), 18 PD98059 (50 µM), 23 PD168393 (100 nM), 24 PP2 (100 nM), 25 rapamycin (10 nM), 26 SB253080 (10 µM), 22 AG490 (40 µM), 27 ZM336372 (1 µM), 28 and nocodazole (50 µM). 29 Next, Alexa Fluor 647-conjugated Tfn and EGFR ligands were allowed to internalize for 5 min, followed by the addition of medium containing unlabeled human Tfn (R&D Systems) and the corresponding EGFR ligands. After the indicated time intervals (0, 5, 30, 60, and 180 min), the cells were fixed by adding an equal volume of methanol-free 4% paraformaldehyde (09154-85, Nacalai Tesque, Kyoto, Japan) for 15 min, washed three times with phosphate-buffered saline (PBS), and subjected to immunofluorescence.