Anti met

Protein interactions were assessed using the Duolink PLA assay (Sigma). Briefly, cells were fixed with 10% neutral buffer formalin, permeabilized with PBS/0.2% Triton X-100 and incubated with the following antibody pairs: anti-CCR2 (1:100 dilution, Biolegend, cat no.357202) and anti-SRC (1:100, Cell Signaling Technology, cat no.2123S), anti-MET (1:50, Santa Cruz Biotechnology, cat no.SC-514148) and anti-SRC, or anti-CCR2 and anti-MET (1:100, Cell Signaling Technology, cat no.4560). Samples were incubated with Duolink in situ Probes; anti-rabbit Minus (cat no.DUO92005) and anti-mouse PLUS (cat no.DUO92001). Signals were amplified with polymerase using In Situ Detection Reagents Green (cat no.DUO92014). Duolink PLA wash buffers (cat no.DUO82049) were used. Cells were counterstained with DAPI. Images were captured at 10x magnification using the FL-Auto EVOS imager.

Cells were lysed using lysis buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X100, 50 mM NaF, 10 mM Na2P4O7, 1 mM Na3VO4, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM PMSF, and a protease inhibitor cocktail). Lysates containing equal amounts of proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio–Rad, Hercules, CA, USA). The membrane was blocked with a 5% skim milk solution and incubated with the following antibodies: anti-ADAR1, anti-CTNNB1, anti-GAPDH, anti-MSI2, anti-MET, anti-SLC38A4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-FLAG-Tag (Cell Signaling Technology, Danvers, MA, USA). An Immobilon™ Western blot detection system (Millipore) was used to detect bound antibodies. The intensities of the western blot bands were quantified using LAS-4000 (Fuji Photo Film Co., Tokyo, Japan).