Anti palk y1604

Co-immunoprecipitation and Western blotting were performed as described previously [16 (link)]. Primary antibodies used included: anti-FOXM1 (1:500, Santa Cruz (SC), #271746, 1:500, Abcam, #207298), anti-pALK Y1604 (1:500, Cell Signaling technologies (CST), #3341S), anti-ALK (1:1000, CST, #3633), anti-pSTAT3 (Y705) (1:2000, CST, #9145), anti-STAT3 (1:1000, CST, #124H6), anti-Cyclin B1 (1:1000, SC, #752), anti-β-Actin (1:8000, SC, #47778), anti-PARP (1:1000, CST, #9542), anti-Caspase 3(1:1000, CST, #9662), anti-Survivin (1:1000, CST, #2808), anti-NPM1 (1:2000, Milipore Sigma, clone 3C9), anti-Vinculin (1:500, SC, #25336), anti HDAC-1 (1:500, SC, #81598), and anti-α-Actinin (1:500, SC, 17829). Secondary antibodies used were HRP-conjugated anti-mouse (1:2000, CST, #7076) and anti-rabbit (1:2000, CST, #7074). Detailed information of western blot can be found at Figure S5.

Total cell lysates were obtained with NP-40 lysis buffer (150 mM sodium chloride, 1.0% NP-40, 50 mM Tris, pH 8.0), supplemented with protease inhibitor cocktail (cOmplete mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein samples were denatured with 2-mercaptoethanol at 95 °C for 5 min. The following antibodies were used for detection: anti-pALK Y1604 (Cell Signaling Technology), anti-ALK (Cell Signaling Technology), anti-pSTAT3Y705 (Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-pERK1/2 (Cell Signaling Technology), anti-ERK1/2 (Cell Signaling Technology), anti-pAkt Y473 (Cell Signaling Technology), anti-AKT, anti-human PARP (Santa Cruz Biotechnology), anti-HDAC8 (H-145;polyclonal; Santa Cruz, Santa Cruz, CA, USA), anti-p-mTOR (Ser2448; Upstate), anti-p-S6K1 (Thr412; Upstate), anti-MET (Cell Signaling Technology), anti-MYCN (Santa Cruz), anti-ac-SMC3 (provided by Prof. K Shirahige, University of Tokyo, Tokyo, Japan) [55 (link)], anti-HSC70 (Santa Cruz), anti-β-actin (clone AC-15; Sigma), anti-actinin (H-2; Santa Cruz) and anti-GAPDH (clone 6C5; Merck).

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 (link)]. Horseradish-peroxidase-conjugated secondary antibodies goat anti-rabbit IgG and goat anti-mouse IgG (1:5,000) were from Thermo Scientific (Waltham, MA).