Fig. 5.

Effect of purified CLPs on interaction of α2β1 integrin with collagen I. (A) Cell adhesion assay was performed as described in Fig. 3 legend using α2K562 cells. Collagen I (10 μg/ml) was immobilized on 96-well plate by overnight incubation at 4°C. Cells were mixed with CLPs at indicated concentrations and pre-incubated at room temperature by 15 min before application on the plate. (B) Inhibition of α2β1integrin binding to immobilized collagen I. Sochicetins were mixed with soluble α2β1 integrin ectodomain (18 μg/ml) and applied on the plate. Collagen-I-bound α2β1 integrin was quantified by ELISA. (C) Direct interaction of sochicetins with recombinant GST-α2A in ELISA. Sochicetins (20 μg/ml) were immobilized on 96-well plate by overnight incubation in TBS, pH 7.4 at 4°C. Plate was blocked with BSA and GST-α2A was incubated on the plate for 1 hour. Detection of bound GST-α2A was performed using polyclonal anti-GST antibody following alkaline phosphatase conjugated anti-rabbit IgG secondary antibody. Plate was read with ELISA plate reader using 405 nm wavelength. Means and SD are shown from experiments performed in duplicates and repeated at least three times