Anti ddr2

Cells were lyzed with RadioImmunoPrecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Villebon sur Yvette, France). Cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. For western blotting, proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, membranes were blocked with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.137 M NaCl, pH 7.4) containing 0.1% Tween (TBS-T) and 5% non-fat dry milk at room temperature during 1 hour and incubated overnight at 4°C with the following primary antibodies: anti-phospho-JAK2 (Santa Cruz Biotechnology), anti-GAPDH, anti-DDR1, anti-phospho-SHP2, anti-SHP2, anti-JAK2, anti-phospho-ERK1/2, anti-ERK1/2, anti-p21^CIP1 (Cell signaling Technology, Saint Quentin Yvelines, France), anti-DDR2 (R&D systems, Lille, France), anti-RAGE (GeneTex, Irvine, CA). Membranes were washed with TBS-T and incubated with the corresponding peroxidase conjugated secondary antibody at room temperature for 1 hour. Chemiluminescent detection was realized by using an ECL Prime Kit (GE Healthcare, Orsay, France).

HT-1080 cells were washed with DPBS and incubated overnight in serum-free media prior 6 hours of stimulation in adult and old type I collagen 3D matrices. Cells were then lysed with RIPA buffer, and cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. Then 300 μg of whole-cell extracts was immunoprecipitated with anti-DDR2 (1:100, Santa Cruz Biotechnology) at 4°C for 12 hours and then bound to protein G agarose beads (GE Healthcare) and finally washed three times with TBS. The proteins were separated by SDS-PAGE, and the immunoprecipitates were blotted with anti-phosphotyrosine 4G10 antibodies (Millipore). The blots were then stripped using a stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 63 mM Tris-HCl pH 6.8) and re-probed with anti-DDR2 (R&D systems) antibody.