Anti pan erk

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).

The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK (for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technology), anti-ALK mAb135 (1:2000; [Witek et al., 2015 (link)]), anti-pERK5 (1:1000; Cell Signaling Technology), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A (1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting; 16B12, Covance). The activating monoclonal antibody mAb46 and ALK monoclonal antibodies mAb13, mAb48 were a kind gift from M. Vigny and have been described previously (Moog-Lutz et al., 2005 (link)). The ALK inhibitor crizotinib was purchased from Chem Express (Shanghai).

BMDMs (10⁶ cells/well) were stimulated with IFNγ (100 U/ml) for the times indicated, lysed and used for Western blot analysis as described previously (30 (link)) with the following adaptations: cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% IGEPAL CA-630 (v/v), 10% glycerol (v/v), 0.1 mM EDTA, 2 mM DTT, 0.2 mM Na-vanadate, 25 mM Na-fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 0.1 μg pepstatin and 1 mM PMSF. The following antibodies were used: anti-IRF1 (Santa Cruz, SC-640), anti-phospho-Tyr701 STAT1, and anti-STAT1 (Cell Signaling Technology, 9167 and 9172), anti-pan-ERK (BD Transduction Laboratories, 610123; p42 is shown in our experiments). Peroxidase-conjugated secondary antibodies (mouse and rabbit) were from Cell Signaling Technology (7076 and 7074). Blots were scanned with a Chemidoc analyzer (Bio-Rad).

FACS-sorted splenic NK cells were lysed in a buffer containing 50 mM Tris/HCl pH 8.0, 10% (v/v) glycerol, 0.1 mM EDTA, 150 mM NaCl (all from Roth), 2 mM DTT, 0.5% NP40 (Igepal CA-630), 25 mM sodium fluoride, 0.2 mM sodium vanadate, 1 mM PMSF, 10 ng/μl leupeptin, 10 ng/μl aprotinin, 10 ng/μl pepstatin (all from Sigma-Aldrich) and lysates were cleared by centrifugation. Ten micrograms protein per lane were loaded and separated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond, GE Healthcare). As a molecular weight standard PageRuler^® Prestained Protein Ladder (Thermo Fisher Scientific) was used. Membranes were probed with anti-STAT1 (Cell Signaling Technology), anti-panERK (BD Transduction Laboratories) and IRDye fluorophor-conjugated secondary antibodies (LI-COR Biosciences). Blots were scanned with Odyssey^® Classic infrared imager (LI-COR Biosciences).