Maintenance and Transfection of HEK293T Cells

HEK293T cells obtained from the American Type Culture Collection were maintained in Dulbecco's modified Eagle's high glucose medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Lonza) in a humidified incubator with 10% CO₂ as described previously30 (link)31 (link)32 (link). Stable HEK293T cells overexpressing GFP-tagged FGFR2 were produced as described previously30 (link). Control and Grb2 knockdown cells were produced as described previously20 (link). Mammalian cell transfection was performed in a six-well plate using Metafectin Pro (Biontx-USA) reagents according to the manufacturer's instructions. Cells were lysed in 50 mM HEPES (pH 7.5), 1% (v/v) Igepal-C630, 1 mg ml⁻¹ bacitracin, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 10% (v/v) glycerol, 50 mM NaCl, 1 mM PMSF and Protease Inhibitor Cocktail Set III (Calbiochem).