Chemidoc imaging station

For Western blot analysis, samples were homogenized (FastPrep FP120, Qbiogene, Illkirch, France) at 10% (weight/volume, w/v) in buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl pH 8.0) and selected samples were digested with proteinase K (PK) (20 μg/mL) (Roche, Mannheim, Germany) for 1 h at 37°C. Digestion was stopped by addition of 10× sample buffer and boiling for 10 min. Samples were analyzed by SDS-page (AnykD, Biorad, Hercules), transferred to PVDF membranes (BioRad) at 400 mA for 1 h, blocked for 1 h at room temperature in protein-free blocking buffer (Thermo Scientific, Rockfort) and incubated overnight at 4°C with anti-PrP antibody Pom1 (1:1000 in blocking buffer; provided by Adriano Aguzzi, Zürich) (43 ). After incubation for 1 h at room temperature with an HRP-conjugated anti-mouse secondary antibody (1:5000 in blocking buffer), signal was detected with ECL femto reagent (Thermo Scientific) and visualized and quantified with a BioRad ChemiDoc imaging station and Biorad VersaDoc. Quantification of relative PrP^Sc amount was performed according to Zhu et al (63 (link)). Briefly, brain homogenate (corresponding to 20 mg total protein) was PK-digested as described above and the relative signal intensity of Pom1-positive signals was measured by the ChemiDoc quantification tool.

Glycerol and NaCl were added to purified DNA, mononucleosomes, or BstXI-digested 12×601 arrays at final concentrations of 40% w/v and 5 mM, respectively, and loaded into the wells of 6% polyacrylamide gels buffered with 0.5x TAE. Nucleic acid-containing samples were separated by electrophoresis at 100 volts for 40 minutes before imaging fluorescence of labeled nucleosomes with a ChemiDoc Imaging Station (BioRad) and staining of DNA using ethidium bromide.

Ba/F3 CD74-ROS1 and CD74-ROS1^D2033N cells were treated with the indicated concentrations of inhibitors for 2 h, pelleted, washed once in ice-cold PBS, and lysed in 200 µL of cell lysis buffer (Cell Signaling Technology) that was supplemented with 0.25% deoxycholate, 0.05% SDS, and protease and phosphatase inhibitors. Equal amounts of protein were extracted with SDS sample buffer for 15 min at 80°C and resolved on 4–15% Tris-glycine or 4–12% Bis-Tris precast gels (Criterion; Bio-Rad). Proteins transferred to Immobilon-FL membranes (Millipore) were probed with: phospho-ROS1 [Cell Signaling Technology (CST); 3078, 1:1000], total ROS1 (CST; 3266, 1:1000), phospho-ERK1/2 (CST; 9101, 1:1000), total ERK2 (Santa Cruz; sc-1647, 1:2000), phospho-AKT (CST; 4060, 1:1000), AKT (BD Transduction Laboratories; 610860, 1:1000), pSHP2 (CST; 3703), pSTAT3 (CST, 9131), and GAPDH (Ambion; AM4300, 1:5000). Blots were imaged using either a LI-COR Odyssey imaging system or the Bio-Rad ChemiDoc imaging station according to the manufacturer’s protocol for immunoblot detection with use of Infrared dye or horseradish peroxidase-conjugated secondary antibodies, respectively.

Lysates were prepared from cells using a standard cell lysis buffer as described before (24 (link)). Protein quantitation of cleared cell lysates was performed using BCA assay, and 25 μg of total protein was loaded on pre-cast 4–12% Bolt™/NuPAGE™ Bis-Tris Protein Gels. Proteins were transferred to nitrocellulose membranes, and probed with relevant antibodies. All antibodies were used at dilutions recommended by manufacturer. Blots were imaged using a Bio-Rad Laboratories (Hercules, CA, USA) ChemiDoc™ imaging station according to the manufacturer’s protocol for immunoblot detection with use of horseradish peroxidase-conjugated secondary antibodies. Spectra Multicolor Broad Range Protein Ladder was used to determine relative molecular weights of protein bands after imaging. Phospho-ROS1 detection required the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher). Densitometry was performed using Image Lab™ Software (RRID:SCR_014210, Bio-Rad Laboratories, Hercules, CA, USA).