PMC

Binding of wild-type and active α1 and α2 I domains to peptide II-27. Experiments were performed as described in the legend to using 0.1 μg of protein/well. Solid-phase adhesion of I domains to peptide II-27 or its shorter derivatives (II-27-A, II-27-B, and II-27-C) is shown. a, adhesion of the wild-type α1 I domain. b, adhesion of the wild-type α2 I domain. c, adhesion of the constitutively active E317W α1 I domain. d, adhesion of the constitutively active E318W α2 I domain. Data are the mean ± S.E. of four independent experiments, each performed in triplicate.

Real-time adhesion of PC12, C2C12-α2⁺, and C2C12-α11⁺ cells to selected peptides. The binding of integrin-expressing cells (25,000 cells/well) was measured using real-time adhesion assay as described under “Experimental Procedures” and in the legend to . Peptides III-7, II-27, II-28, and GVOGEA were used as adhesive coatings, with GPP10 as the control. The cell line and integrin expressed are indicated above each panel, which shows the mean cell index after 2 h ± S.E. from triplicate determinations from each of three experiments.

Equilibrium binding of α1 I domain to collagenous substrates. a, inhibition of binding of the wild-type α1 I domain to collagen IV (Col-IV) was determined as described in the legend to in the presence of peptides. The I domain was preincubated with the indicated amount of GLOGEN, GFOGER, and GPP10. Data show the mean ± S.E. of three experiments. b, adhesion of the wild-type α1 I domain to GLOGEN, GFOGER, GVOGEA, and GPP10 was measured using the SRU Biosystems BIND Explorer as described under “Experimental Procedures”, allowing K_D to be estimated. ΔPWV, shift in peak wavelength value.

Real-time binding of PC12 cells to II-27 and shorter II-27-derived peptides. E-Plate^TM wells were coated with the indicated peptides as described in the legend to (c and d). The sequences of the peptides are shown above the graph. Cell adhesion was monitored for 2 h, and data were collected every minute. Every fifth data point is shown. Experiments were performed in triplicate in the presence of either 2 mm Mg²⁺ or EDTA as indicated, and the mean cell index ± S.E. is shown from four independent experiments.

Binding of Rugli cells to selected Toolkit peptides before and after activation by PMA. Rugli cells were applied to peptide-coated E-Plates^TM prepared as described under “Experimental Procedures,” and the cell index was recorded at 1-min intervals. For clarity, fewer time points are shown in each graph. The mean cell index ± S.D. is shown from one representative experiment, performed in triplicate, from three similar data sets. The peptides used are indicated. Traces close to the base line include adhesion to the GPP10 control and to the indicated peptides in the presence of EDTA. a, adhesion of control Rugli cells. b, adhesion of Rugli cells activated, in parallel with controls, for 30 min with 1 μm PMA.

Effect of anti-integrin antibodies on PC12 cell binding to peptide III-7. Cells were incubated with anti-α1 or anti-α2 antibodies or controls as indicated for 30 min. Mg²⁺-dependent label-free binding of PC12 cells was then measured as described under “Experimental Procedures” and in the legend to (c and d). a, time course of adhesion measured at 10-min intervals over 2 h expressed as the mean cell index ± S.E. of three experiments. b, inhibition of PC12 cell adhesion to peptide III-7 in the presence of increasing levels of anti-α1 antibody shown as end point determinations after 2 h. Data are the mean cell index ± S.E. of five independent experiments. Where not visible, errors lie within the dimensions of the symbols.

Binding of wild-type α1 I domain to Toolkits II and III and to selected shorter triple-helical peptides. The recombinant wild-type α1 I domain was incubated in wells of Immulon 2HB 96-well plates coated with peptides, and adhesion was measured in the presence of either 2 mm Mg²⁺ (black bars) or 2 mm EDTA (white bars) as described under “Experimental Procedures.” BSA, GPP10, and GFOGER served as control surface coatings. Experiments were performed in triplicate using 0.1 μg of protein/well. a, adhesion to Toolkit II; b, adhesion to Toolkit III; c, adhesion to selected shorter peptides, formatted with the indicated sequence flanked by (GPP)₅ host peptides. Long GFPGER has the same sequence as II-28 but with “O” in its GFOGER motif replaced with “P.” Data are the mean ± S.E. of at least six independent experiments. Col, collagen.

Binding of PC12 cells to Toolkits II and III and selected peptides. Immulon 2HB 96-well plates or E-Plates^TM for the xCELLigence instrument were coated with the indicated peptides as described under “Experimental Procedures.” a and b, PC12 cells were seeded at 10⁵/well, and SPBA was performed after 1 h as described using the colorimetric lactate dehydrogenase assay kit. c and d, PC12 cells were seeded onto E-Plates at 2.5 × 10⁴ cells/well, and the cell index was measured continually. PC12 cell binding to Toolkits II (a) and III (b) is shown. PC12 cell adhesion to the indicated Toolkit peptides (c) and to selected shorter integrin-binding peptides (d) is shown as end point data after 2 h of adhesion. All experiments were performed in triplicate in the presence of either 2 mm Mg²⁺ or EDTA as indicated. Data are the mean ± S.E. of three independent experiments.