Odyssey imaging system

Where indicated, cells were treated with tyrosine kinase inhibitor for 2 to 4 hours. Lysates were prepared from cells using a standard cell lysis buffer as described before (21 (link)). Protein quantitation of cleared cell lysates was performed using Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific), and 25 μg of total protein was loaded on pre-cast 4–12% Criterion™ XT Bis-Tris Protein Gels (Bio-Rad, # 3450125). Proteins were transferred to nitrocellulose membranes, and probed with phospho-ROS1 [#3078, 1:1,000; Cell Signaling Technology (CST)], total ROS1 (#3266, 1:1,000; CST), phospho-SHP2 (#3751, 1:1000, CST), total SHP2 (#3397, 1:1000; CST), phospho-ERK1/2 (#9101, 1:1,000; CST), total ERK2 (sc-1647, 1:2,000; Santa Cruz, phospho-Akt (#4060, 1:1,000; CST), AKT (#610860, 1:1,000; BD Transduction Laboratories). 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. phospho-ROS1 detection required the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific).

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.