Cell culture, lysis, and immunoblotting

PC12 cells were transfected with different ALK constructs by electroporation. Cells were serum-starved for 36 hours prior to stimulation with 1 μg/ml of the activating mAb46 for 30 minutes Cells were washed with ice-cold PBS prior to harvest in lysis buffer [25 mM of Tris-Cl, pH7.5, 150 mM of NaCl, 1% (v/v) Triton X-100, 1 mM of DTT, protease inhibitor cocktail tablet (Roche)]. Cell lysates were cleared by centrifugation at 14,000 rpm for 15 minutes at 4°C. Samples were boiled in 1x SDS sample buffer and analyzed by immunoblotting. Primary antibodies used for immunoblotting were: anti-pan-ERK (1:10,000) from BD Transduction Laboratories (Franklin Lakes, NJ); anti-pALK (Y1604) and anti-pERK1/2 (T202/Y204) from Cell Signaling Technology (Danvers, MA); anti-FAM150A and anti-FAM150B antibodies from Atlas Antibodies (Stockholm). Monoclonal antibody 135 (anti-ALK) was produced in the Hallberg laboratory against the extracellular domain of ALK as described [20]. Horseradish-peroxidase-conjugated secondary antibodies goat anti-rabbit IgG and goat anti-mouse IgG (1:5,000) were from Thermo Scientific (Waltham, MA).