Enzyme free detachment medium

For the co-immunoprecipitation and collagen II-induced signaling experiments, MXF8000 cells were detached using enzyme-free detachment medium (Gibco) and kept in suspension in serum-free medium with 2% BSA for 30 min, then replated on dishes pre-coated with collagen II (Millipore) at 10 μg/ml. Cells were harvested three hours later (for immunoprecipitations) or at the times indicated in the figure. For immunoprecipitations, total lysates were prepared in lysis buffer (20 mM Tris-HCL, 150 mM NaCl, 1% Triton-X100, 5 mM MgCl₂, 1 mM EDTA, proteinase inhibitor cocktail [SIGMA], and phosSTOP tablet [Roche]). Lysate was subjected to immunoprecipitation using anti-integrin-α10 (AB6030) or rabbit IgG (SIGMA), then immunocomplexes were subjected to Western blotting using anti-integrin-α10 monoclonal antibody (Neobio).
GTP-RAC was assessed using a GST-PBD pulldown assay as described (60 (link)).

Cells were detached using enzyme-free detachment medium (Gibco), then resuspended in serum-free medium with 2% BSA. Fifteen thousand cells were replated on 96-well plates pre-coated with 1% BSA, collagen I, or collagen II (10 μg/ml) for 50 min. The plates were washed, then attached cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. After overnight treatment with 0.5% Triton-X100, optical density of the plates was read at 595 nm.