Phospho ddr2 y740

All media and FBS were purchased from Thermo Fisher Scientific (Waltham, MA); proMMP-13 enzyme-linked immunosorbent assay (ELISA) kits from R&D systems (Minneapolis, MN); Phosphatase Inhibitor Cocktail [#5870; Cell signaling, Danvers, MA, USA] and protease inhibitor cocktail (#539131, Calbiochem, San Diego, CA, USA); The Wnt signaling inhibitor CCT031374 hydrobromide (cat. no. 4675/10, R&D Systems, Minneapolis, MN), antibodies to β-Catenin (D10A8; XP® Rabbit mAb #8480), α-Tubulin (11H10; Rabbit mAb#2125), Akt (#9272); Phospho-Akt (Ser473) (193H12; Rabbit mAb #4058), Phospho-DDR1 (Tyr792; #11994) and total DDR1 (D1G6; XP® Rabbit mAb #5583) from Cell Signaling Technology (Danvers, MA 01923); the antibodies to human Phospho-DDR2 (Y740; #1119D) and human DDR1 (AF2538) from R&D system (Minneapolis, MN); HRP-conjugated rabbit anti-goat (#305-036-047), HRP-conjugated goat anti-rabbit (#111-035-144), AffiniPure goat anti-human lgG, F(ab’)2 fragment specific (#109-005-097) and peroxidase affinipure goat anti-human lgG(H+L; #109-035-088) from Jackson ImmunoResearch (West Grove, PA, USA). Human DDR1-full length [19 (link)] and DDR1-Fc [20 (link)] were kind gifts from Dr. Friedemann Kiefer, Max Planck Institute for Molecular Biomedicine, Germany, and from Birgit Leitinger, National Heart and Lung Institute, Imperial College London, London UK, respectively.

Total protein concentration was determined with the BCA Protein Assay Kit (Beyotime). The protein extracts were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Nonspecific protein binding was achieved by incubation of the membranes with 5% dried skim milk for 1 h, and the membranes were incubated overnight with antibodies against CARM1 (1 : 1000, Abcam), histone H3R17 dimethylation (1 : 800 Millipore), p15 (1 : 1000, Abcam), p16 (1 : 1000, Abcam), p21 (1 : 800, Abcam), TERT (1 : 500, Abcam), DDR2 (1 : 500, R&D), phospho-DDR2 Y740 (1 : 500, R&D), and β-actin (1 : 1000, ABclonal). The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 5000, ABGENT) for 1 h. The results were visualised by the Chemiluminescent HRP Substrate (Millipore) and recorded by a Bioshine ChemiQ imaging scanner (Bioshine).