Bio1211

We used commercially available human pCRP (Lee BioSolutions, St Louis, MO, synthesized in E.Coli). pCRP was stored in 10 mM Tris-HCl (pH 7.5) with 2 mM CaCl₂ to prevent spontaneous formation of mCRP from pCRP. mCRP was prepared by treating pCRP with 8 M urea/10 mM EDTA for 1 h at 37°C as described [14] (link), [15] (link). We did not detect endotoxin in the pCRP used in this study using endotoxin detection kit (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific) (data not shown). mAb 7E3 (anti-human integrin β3) and mAb AIIB2 (anti-human integrin β1) hybridomas were obtained from ATCC. mAb SG73 (anti-human α4) hybridoma was a kind gift from K. Miyake (University of Tokyo). Anti-phospho-AKT (Thr-308), anti-phospho-ERK1/2, anti-ERK1/2, anti-AKT were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Cyclic RGDfV [16] (link) was purchased from Enzo Life Sciences (Plymouth Meeting, PA). BIO1211 was obtained from Tocris Bioscience (Ellisville, MO). LY294002 and PD98059 were purchased from Promega (Madison, WI). Chinese hamster ovary (CHO) cells that express WT β1, β3, or the β1–3-1 mutant have been described [17] (link). CHO cells that express human α4 have been described [18] (link). Recombinant soluble αvβ3 has been described [19] (link).

BIO1211 and BIO5192, both integrin α4β1 (VLA‐4)‐specific inhibitors, were purchased from Tocris Bioscience (Bristol, UK). BIO1211 was reconstituted in PBS and BIO5192 was in DMSO.