DDR Autophosphorylation Assay

The assay was performed as described before [42]. Briefly, DDR-expressing HEK293 cells or empty-vector control HEK293 cells were grown in 12-well plates. 24 h later, the cells were incubated with serum-free medium for 16 h. Cells were then stimulated with collagen I (at 10 µg/ml) or different collagen peptides (at 100 µg/ml) for 90 min, at 37°C. In the inhibition experiment, cells were pre-incubated with dasatinib for 30 min at 37°C prior to collagen stimulation. Cells were lysed in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, 5 mM NaF. Aliquots of the lysates were analyzed by SDS-PAGE followed by blotting onto nitrocellulose membranes. The duplicate blots were probed with either anti-phosphotyrosine mAb or anti-DDR Abs followed by corresponding secondary Abs. Detection was performed using Enhanced Chemiluminescence Plus (Amersham Biosciences) on an Ettan DIGE Imager (GE Healthcare Biosciences).