<p>Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. a<u>) ELISA-type integrin binding assay.</u> In a) wells were incubated with recombinant soluble αvβ3 for 2 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl<sub>2</sub>. b) <u>Effect of heat treatment on mCRP binding to soluble αvβ3.</u> We heated (90°C for 20 min) mCRP before coating wells and used for binding assays. Assays was performed as in a). c) <u>β3- and β1-3-1-CHO cells adhere to mCRP, but β1-CHO cells did not adhere well to mCRP.</u> mCRP was incubated with β1-CHO, β3-CHO, or β1-3-1-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl<sub>2</sub>. Bound cells were quantified. d) <u>Specificity of αvβ3 binding to mCRP.</u> We tested if inhibitors of αvβ3 block adhesion of β3-CHO cells to mCRP. mCRP was incubated with cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl<sub>2</sub>. mAb 7E3 (to human β3, 10 μg/ml) and cyclic RGDfV (specific antagonist to αvβ3, 10 μM) blocked the adhesion of β3-CHO cells to mCRP, but control purified mouse IgG (mIgG) or vehicle DMSO did not. e) <u>Cation dependency of mCRP binding to αvβ3</u>. Adhesion assays were performed as described in c). mCRP was incubated with β3-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 2 mM cations or EDTA. The coating concentration of mCRP is 50 μg/ml. The levels of adhesion in different cation conditions are statistically different. f) Alignment of β1, β3, and β1-3-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093738#pone.0093738-Takagi1" target="_blank">[17]</a>. g) <u>Specificity of β1-3-1 integrin binding to mCRP.</u> We tested if anti-human β1 mAb AIIB2 blocks β1-3-1-CHO cells adhesion to mCRP. (Note: 99% of β1-3-1 is β1 and mAb AIIB2 binds to β1-3-1 and blocks its function). h) <u>Cell adhesion to pCRP. </u>pCRP was incubated with β1-CHO, β3-CHO, or β1-3-1-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl<sub>2</sub>. Bound cells were quantified.</p
