Luminex assays

The Bio-Plex Pro Human Cytokine 27-plex Assay (#M500KCAF0Y, Bio-Rad) was used to measure levels of cytokines associated with varied types of immune response, while individually coupled magnetic beads were used to detect growth factors associated with implicated ligand-RTK interactions from our computational coexpression analysis (Fig. 1, E and G). To generate our own immunoassays, we used capture and detection antibodies against human neuregulin 1 beta 1, GAS6, HGF, EGF, amphiregulin, heparin-binding EGF, VEGF, and transforming growth factor–α purchased as DuoSets from R&D Systems. For antibody-bead coupling, Bio-Plex Pro magnetic COOH beads (Bio-Rad) were activated with EDC [N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; Sigma-Aldrich] and sulfo-NHS (N-hydroxysulfosuccinide; Pierce) in 100 mM phosphate buffer (pH 6.3) for 20-min shaking at room temperature and then incubated with capture antibody (0.1 mg/ml) in 50 mM Hepes buffer (pH 7.4) overnight shaking at 4°C. The following day, beads were washed 3× in 1% bovine serum albumin (BSA) in PBS and then stored at 4°C. Immunoassays were adapted to a 384-well plate format, performed on 1× or 1/10× dilutions of clarified supernatants according to the manufacturers’ protocols, and read on a FLEXMAP 3D system (Luminex Corp). For each analyte, five-parameter logistic regression was used to fit standard curves for absolute quantification. Measured levels of analytes were normalized on the basis of the micro bicinchoninic acid protein assay (Pierce), and the Benjamini-Hochberg procedure was used to correct for multiple comparisons between treatment conditions.

A custom Bio-Plex phosphoprotein panel was used to measure levels of intracellular kinases and consisted of immunoassays detecting phosphorylated versions of 11 key signaling nodes: cJun (pSer⁶³), JNK (pThr¹⁸³/pTyr¹⁸⁵), p70 S6K (pThr⁴²¹/pSer⁴²⁴), MEK1 (pSer²¹⁷/pSer²²¹), signal transducers and activators of transcription 1 (Stat1) (pTyr⁷⁰¹), Akt (pSer⁴⁷³), ERK1/2 (pThr²⁰²/pTyr²⁰⁴ and Thr¹⁸⁵/pTyr¹⁸⁷), p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²), nuclear factor κB p65 (pSer⁵³⁶), Stat3 (pTyr⁷⁰⁵), and glycogen synthase kinase 3a/b (pSer²¹/pSer⁹). R&D DuoSets against human AXL, MERTK, MET, and CSF1R were used to generate immunoassays against these RTKs. For RTK pTyr measurements, an anti–pan-pTyr antibody (4G10, Sigma-Aldrich) was used in place of the RTKs’ corresponding detection antibodies. Bio-Plex cell lysis buffer (Bio-Rad) was used to lyse cells for intracellular kinase measurements, while NP40 cell buffer [20 mM tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and 10% glycerol (pH 7.4)] was used for RTK measurements. Lysis buffers contained protease and phosphatase inhibitors per manufacturer guidelines (Bio-Rad). In both cases, immunoassays were performed on clarified lysates diluted twofold in assay buffer consisting of 1% BSA in PBS.