The following antibodies were used: antibody against human β1 integrin, isotype antibody control IgG2a,κ, anti-FN, anti-tensin (all from BD Biosciences), anti-human integrin-α5 nonfunction-blocking mAb11 [26 (link)], anti-human integrin-α5 inhibitory mAb16 [27 (link)], anti-human integrin-β1 inhibitory mAb13 [28 (link)], anti-human integrin-αV L230 (ATCC), anti-human FN mAb 13G12 [28 (link)], anti-β3 integrin (sc-7311; Santa Cruz Biotechnologies), anti-phospho-FAK (Fischer Scientific). Other antibodies used were from Sigma-Aldrich: anti-actin, anti-talin, anti-α-actinin, anti-vinculin and anti-paxillin. Secondary species–specific FITC-, Cy3- or AMCA-conjugated antibodies were from Jackson ImmunoResearch Laboratories. Rhodamine-phalloidin was from Fischer Scientific.
Human plasma FN was purified according to Miekka [29 (link)] and 70kD fibronectin fragment was obtained as described [30 (link)]. The 120kD fragment from plasma FN was purchased from Merck. Fibronectin-depleted FBS was obtained by the use of Gelatin-Sepharose 4B (LKB) as described by Knox [31 (link)]. Cellular fibronectin was purified according to Yamada et al. [32 (link)]. HiLyte Fluor™ 488 labeled bovine FN was purchased from Cytoskeleton Inc.
Human plasma FN was purified according to Miekka [29 (link)] and 70kD fibronectin fragment was obtained as described [30 (link)]. The 120kD fragment from plasma FN was purchased from Merck. Fibronectin-depleted FBS was obtained by the use of Gelatin-Sepharose 4B (LKB) as described by Knox [31 (link)]. Cellular fibronectin was purified according to Yamada et al. [32 (link)]. HiLyte Fluor™ 488 labeled bovine FN was purchased from Cytoskeleton Inc.