Total erk2

LR73 phagocytes or BMDMs were seeded in 10 cm plates at a concentration of 2×10⁶ cells per plate. In some experiments, apoptotic Jurkat cells were added to LR73 cells at a 10:1 ratio and incubated for the indicated times. After washing unbound Jurkat cells, the remaining cells were lysed in RIPA buffer and used in total protein Western blots or phosphorylation blots (Phos-Tag; Wako). The blots were probed using SLC12A2 (Cell Signaling Technology), SLC12A4 (Abcam), and total ERK2 (Santa Cruz Biotechnology, #sc-154-G) antibodies in Can Get Signal solution (TOYOBO) followed by chemiluminescence detection.

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.