The surfaces were then coated with 1% fatty acid-poor bovine serum albumin (BSA, Sigma, St. Louis, MO) for 40 min at 37uC and washed with PBS. Cells, which had been suspended from confluent monolayers by trypsinization followed by inhibition of trypsin with fetal calf serum and repeated centrifugation alternating with suspension in PBS to remove serum proteins, were seeded in the coated wells at 37uC in Dulbecco’s modification of Eagle’s medium (DMEM, Cellgro Mediatech, VA) supplemented with 0.2% fatty acid-poor BSA. The number of cells added was determined in pilot experiments to result in monolayers of spread cells 60,70% confluent after 4 hr. Preparation of fibronectin and proteolytic fragments of fibronectin Human fibronectin was purified from a fibronectin-rich side fraction of plasma processing by precipitation of contaminating fibrinogen by brief heating at 56uC followed by chromatography on DEAE-cellulose [23]. A schematic of the fibronectin proteolytic fragments and recombinant constructs are shown in Fig. 1. ‘‘Fingerless’’ fibronectin was prepared by reduction and alkylation, limited trypsin treatment, and gel filtration [24]. The method is based on the finding that disulfide modification greatly increases the susceptibility of N- and C-terminal type I and II modules to trypsinization [25]. A mixture of fragments was obtained [24]. This mixture was dialyzed against buffer A (25 mM Tris pH 7.4, 50 mM NaCl) and applied to an anionic exchange column (monoQ HR 5/5, GE Healthcare, Piscataway, NJ) in an Akta FPLC (GE Healthcare). Proteins were separated by linear gradient of buffer B (25 mM Tris pH 7.4, 1 M NaCl) and characterized by immunoblotting with previously characterized monoclonal antibodies L8, 9D2, C4B10, or IST-7 [26] or antibodies 4D1 or 5C3 generated at the University of Alabama- Birmingham [27]. For the immunoblotting, peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, PA) was used as a secondary antibody. Gelatin-binding 160/180-kDa fibronectin fragments were purified from a limited tryptic digestion of native plasma-derived fibronectin [28]. The C-terminal 31-kDa fragment was purified from the non-gelatin-binding fraction of the same limited tryptic digest [29]. The N-terminal 70-kDa catheptic D fragment of fibronectin was purified as described previously [24]. Recombinant proteins Proteins are named according to their N- and C-termini with the nomenclature of Campbell et. al. [30]. Also included in the name are ‘‘A’’ if AF3 was present and the version of the V-region [31–35]. Bacterially synthesized recombinant proteins 7F3-10F3, 12F3-14F3, 7F3-14F3, 7F3-14F3A, 2F3-11F3, and 2F3-14F3 (Fig. 1B) were purified and characterized as described [9,36,37]. Recombinant proteins 1F1-3F3, 6F1-C V89, 6F1-CA V89, 1F3-C V89, 1F3-CA V89, 1F3-14F3, 2F3-C V89, 1F3-C V0, 1F3-CA V0, 1F3-C V64, 1F3-CA V64, 1F3-C D(V15F310F1), and 1F3-CA D(V15F310F1) (Fig. 1B) were produced using a baculovirus expression system [38]. Recombinant baculovirus was generated by co-transfection of monolayers of SF9 cells (Invitrogen, Carlsbad, CA) with BaculoGold linearized AcNPV viral DNA (BD Biosci- ences, San Jose, CA) and the appropriate transfer vector using CellFectin (Invitrogen). The transfer vectors were constructed by cloning PCR-amplified pieces of DNA of human fibronectin into pCOCO; pCOCO endows the recombinant protein with an N- terminal signal peptide and C-terminal polyhistidine tag [38]. Viruses from individual plaques were amplified, and used after pass Self-Help Fibronectin Assembly 3 to infect High Five insect cells (Invitrogen) in SF900II serum-free medium at 27uC. Sodium azide, 0.1%, and polymethylsulfonyl fluoride, 2 mM, were added to conditioned medium collected ,65 hours post infection. The medium was dialyzed against TBS (10 mM Tris, pH 7.4, 130 mM NaCl) and then incubated with a suspension of Ni2+-nitriloacetic acid resin (Qiagen, Valencia, CA) overnight at room temperature. The resin was collected by centrifugation, placed in a small column, and washed with TBS buffer containing 15 mM imidazole. Polyhistidine-bearing protein was eluted with TBS buffer containing 300 mM imidazole, and the purified protein, 200–1000 mg/ml, was dialyzed against Tris buffer containing 1 M sodium bromide, and stored in portions at 280uC until used. Alternatively, MOPS buffer (10 mM MOPS, pH 7.4, 300 mM NaCl) is used instead of TBS buffer. Fluorescent labeling of fibronectin and 70-kDa fragment Fibronectin or N-terminal 70-kDa fragment was labeled with Rhodamine RedTM-X (FluoReporter Rhodamine RedTM-X (RX) Protein Labeling Kit, Invitrogen) according to the manufacturer’s instructions with a slight modification. RX was dissolved in DMSO and diluted in 0.5 M carbonate buffer, pH. 9.5, to a final concentration of 0.5 mg/ml, and fibronectin or the 70-kDa fragment were at a concentration of 2 mg/ml. Alternatively, fibronectin or the fragment was labeled with fluorescein isothiocyanate (FITC) as described [24]. Fluorescence microscopy Labeled fibronectin or 70-kDa fragment, 9 or 2.7 mg/ml (approximately 40 nM), was added to the culture medium at the time of addition of fibronectin-null cells to cover slips coated with various proteins. After 4-hr incubation at 37uC, cells were washed thrice and fixed with 3.7% paraformaldehyde for 15 min, followed by washing with PBS. Alternatively, cells were allowed to adhere for 2 hr and then incubated for an additional 2 hr with fibronectin or 70-kDa fragment before washing and fixation, or adhered for 1 hr and incubated for an additional 3 hr. Results were reproducible for differently labeled fibronectin (Rhodamine- or FITC-labeled fibronectin) added for slightly different durations by three different scientists over 10 year’s period. The difference between different substrates within each figure was always consistent, although differently labeled fibronectin caused some difference between different figures. In other experiments, cells were also incubated with or without 500 nM lysophosphatidic acid (LPA) or 10 mg/ml 9EG7 (anti-mouse b1 integrin, Pharmingen, CA) during incubation. For dual fluorescence of FITC-fibronectin and rhodamine labeled actin, vinculin, or b1 integrin, cells were fixed and permeabilized with 3.7% paraformaldehyde for 15 min and 0.2% Triton X-100 for 5 min, using Rhodamine-phalloidin (Sigma, St. Louis, MO), monoclonal antibody against vinculin (Sigma v-9131), or 9EG7 against ß1 integrin followed by RX conjugated AffiniPure donkey anti-mouse or goat anti-rat IgG (Jackson ImmunoResearch). Cover slips were mounted on Vectashield (Vector laboratories, Burlingame, CA) and viewed on an Olympus epifluorescence microscope (BX60, Olympus America Inc., Melville, NY). Pictures were taken with RT Slider digital camera (Spot Diagnostic Instruments, Inc., Sterling Heights, MI) and processed with Spot RT Software v3 and Adobe Photoshop (Adobe System Inc., San Jose, CA). Quantification of bound or deposited fibronectin, bound 70-kDa fragment, and other proteins After incubation of fibronectin-null cells in DMEM containing 0.2% BSA and 40 nM RX-fibronectin, FITC-fibronectin, or PLoS ONE | www.plosone.org 3 January 2009 | Volume 4 | Issue 1 | e4113