Image studio lite software version 4

Cells were lysed using radioimmunoprecipitation assay buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) with protease inhibitor cocktail (Sigma-Aldrich). The protein concentration in the cell lysates was measured with a bicinchoninic acid assay (Bio-Rad Laboratories). 30 µg of each cell lysate was run in an SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking in 4% nonfat dry milk in TBS-T solution for 1 h, the membrane was first incubated with the primary antibody at 4°C overnight, then incubated with the secondary antibody for 1 h at RT. The membranes were imaged using an Odyssey CLx imaging system (LI-COR Biosciences). For phosphorylated PDGFRα WB, CAFs and NFs were starved overnight and, the next morning, were stimulated with complete culture medium for 2 h. Cell lysates were then prepared with addition of PhosSTOP phosphatase inhibitor cocktail (MilliporeSigma) to the lysis buffer. HRP-conjugated secondary antibodies were used and detected with SuperSignal West Femto maximum sensitivity substrate kit (Thermo Fisher Scientific) via an Amersham Imager 600 (GE Healthcare). The normalized band intensities were measured with Image Studio Lite Software version 4 (LI-COR Biosciences), which were further normalized to loading controls, either β-actin or α-tubulin.