MIA protein was denatured at 70°C for 10 min after addition of reducing and denaturing Roti-Load buffer (Roth, Karlsruhe, Germany) and subsequently separated on SDS 12.75% polyacrylamide gels (Invitrogen, Groningen, The Netherlands). After transferring the proteins onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), the membrane was blocked using 3% BSA in PBS for 1 h at room temperature (RT) and incubated with a 1:150 dilution of primary polyclonal rabbit anti-MIA antibody (Biogenes, Berlin, Germany) in 3% BSA/PBS overnight at 4°C. After washing in PBS, the membrane was incubated with a 1:2000 dilution of an alkaline-phosphate–coupled secondary antibody (Chemikon, Hofheim, Germany) for 2 h at RT. Finally, after washing steps, immunoreactions were visualized by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) (Invitrogen) staining.Luminescent labeling of human MIA protein
Human MIA protein (100 µg) was labeled with Ru(bpy)3-isothiocyanate (1 mg) (Cat. no. 15412; Active Motif Chromeon, Tegernheim, Germany) in 640 µL bicarbonate buffer (pH 9.3) supplemented with 200 µL DMSO required for dissolving the dye. After 50 min, the reaction mixture was purified on a size-exclusion column (Sephadex G-25 M PD-10 desalting column; Amersham Pharmacia Biotech, Uppsala, Sweden) and samples of the collected fractions as well as a dilution series of unlabeled MIA protein were analyzed by Western blotting as described in the “Protein analysis by Western blotting” section, above.Biotin conjugation of peptide AR54
0.25 mg of AR54 was dissolved in 30 µL bicarbonate buffer (pH 9.3). After addition of 0.38 mg biotin-N-hydroxy succinimide (biotin-NHS) (Cat. no. 203188; Calbiochem, Gibbstown, NJ, USA) in 10 µL DMSO, the reaction mixture was incubated overnight at 4°C. As the NHS-ester was expected to be completely reacted or hydrolyzed, no purification was carried out.Coating of well plates with AR54-biotin and MIA-biotin
Black streptavidin-coated 96-well plates (Cat. no. 655997, Greiner Bio-One, Frickenhausen, Germany), with 20 pmol streptavidin loaded per well, were treated with 20 equivalents AR54-biotin per mol of (tetrameric) streptavidin in PBS (pH 7.4). An uncoated control lane was sealed with adhesive film to prevent contamination with AR54-biotin. After addition of AR54-biotin, the entire plate was sealed with adhesive film and incubated for 3 h under agitation. The coated lanes were washed five times with PBS (pH 7.4) before being air-dried and sealed with adhesive film, which was removed only immediately before use of each lane.
MIA-biotin was prepared as previously reported (6) and used for treating a well plate as described in this section, except that the plate was not dried and used for measurements immediately.Polarization assay setup
All measurements were performed at RT on a Polarstar Optima microplate reader (BMG Labtech, Offenburg, Germany). A 390–10-nm bandpass filter was used for excitation while a 520-nm longpass filter was used for the emission light. Even though the extinction coefficient is higher at longer wavelengths, we chose a shorter excitation wavelength as this led to higher polarization values. A MIA-Ru(bpy)3 concentration of 55 fM was used in all experiments. A solution volume of 250 µL/well was found to give a low standard deviation with high signal intensity. Unless otherwise indicated, all measurements were performed in PBS without calcium or magnesium (PAN Biotech GmbH, Aidenbach, Germany). Addition of components to the wells was done in the following order: interaction partner, buffer, and MIA-Ru(bpy)3. Owing to different reaction kinetics, measurements were performed every 5 min over a 30-min period. Polarization values are reported relative (P/P0) to the value of free MIA-Ru(bpy)3 in solution in a well not treated with AR54-biotin. All reported values are an average of three independent measurements.Results
In previous studies, FN14—a peptide that matches a fibronectin domain—was identified as an MIA protein binder in a phage display experiment (7,22). AR54, a MIA protein–binding peptide deduced from peptide FN14, has been shown by Boyden Chamber invasion assay to functionally inhibit MIA protein in vitro. The peptide was able to almost completely inhibit MIA protein function by preventing interactions of MIA protein with extra-cellular matrix molecules and integrins, without affecting cell migration itself (23). Using AR54 as a known inhibitor of MIA protein, we aimed to establish an assay which allows detection of potential MIA protein–inhibitory compounds.