293T (American Type Culture Collection), U2OS, and RCS cells (obtained from B. de Crombrugghe) (74) were propagated in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and antibiotics (Invitrogen). All expression vectors are listed in table S1. To obtain cells stably expressing FGFR4-GFP, U2OS cells were transfected with pEGFP-N1-FGFR4 (75) using FuGENE6 according to the manufacturer’s protocol (Promega). Clones were selected with geneticin (1 mg/ml), and their FGFR4-GFP expression amounts were analyzed by immunofluorescence and Western blot. A homogenous clone with moderate FGFR4-GFP expression was chosen for further studies. U2OS cells stably expressing FGFR1-GFP and FGFR1-BirA-HA have been described (76). For generation of truncated SHIP2 variants, the C-terminal Myc-DDK tag in pCMV6-hSHIP2 vector (OriGene) was replaced by V5-HIS tag using a NEBuilder HiFi DNA assembly kit (New England Biolabs). Truncated human SHIP2 constructs were generated by polymerase chain reaction (PCR) mutagenesis. Catalytically inactive 5′-PD SHIP2 was created by introducing P686A, D690A, and R691A substitutions into the inositol phosphatase domain by site-directed mutagenesis (Agilent). For cell migration assay, cells were plated on fibronectin-coated eight-chambered μ-slide wells and allowed to migrate for 15 hours as reported before (23, 77). Cells were analyzed with a Leica DM6000B microscope, using 10× magnification objective for live-cell imaging. Each cell was tracked over the period of time using the manual tracking plugin of the ImageJ software. FGF2, NGF, and EGF were from R&D Systems, heparin was from Sigma-Aldrich, AZM475271, A419259, and AS1949490 were from Tocris Bioscience.

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