Abts solution

A 96-well plate was coated with the cytoplasmic domain of ADAM15 (100 ng/100 μl in PBS) blocked with 1% BSA in PBS for 1h, and incubated with the GST-tagged PABP domains (500 nM) for 1h at room temperature. After three washes with PBS/ 1% Triton X-100, wells were incubated with HRP-conjugated anti-GST antibodies (1:500; Sigma-Aldrich) for 1h, developed with 100μl ABTS solution (Roche, Mannheim, Germany) and the optical densitometry (OD) read at 405 nm.

All solutions used in ELISA were made up in 1% w/v BSA in PBS and all incubations were carried out at room temperature on the shaker unless otherwise stated. PBS-T was used for all washing steps. Coupling of BSA-conjugated BgAgs (pentameric sugars from IsoSep AB, Sweden) to 96 well plates (NUNC Immobilizer Amino) was carried out by adding 100 μl of a 5 μg/ml sugar solution in sodium carbonate buffer to each well. Control wells contained BSA in PBS buffer at 100 μg/ml. Plates were incubated for 2 h before removing the solutions and washing three times. All wells were blocked by addition of 100 μl of 100 μg/ml BSA in PBS buffer and incubation, as before. Plates were emptied and tapped dry. Digoxigenin tagged bacteria were diluted to an OD₆₀₀ of 0.05 and pre-incubated with QPLEX (34 μM) or not (control). 100 μl of bacterial solution was added to each well and plates were incubated overnight. Plates were washed three times (ELISA-washer was used for three washes with 1 min interval) before adding 100 μl anti-digoxigenin-POD solution (1 in 5,000, Roche Diagnostics) to each well and incubating for 1 h. Plates were washed five times and tapped dry before developing in ABTS solution (Roche Diagnostics) and were read at 405 nm. Specific binding was determined by subtracting the binding to BSA from the binding to the BgAgs for each strain.

Cells were seeded in 96-well plates at a concentration of 4x10⁴ cells per well. AS and ASs6 cells were transfected with a VEGFR-2 expression vector using FuGene (Promega) according to the manufacturer's protocol. On the next day cells were serum-starved for 24 h. After starvation cells were washed twice with PBS and blocked for 1 h at room temperature with 100 μl/well of PBS plus 5% BSA. Where indicated, cells were treated with 100 ng/ml rat v6 peptide or 100 ng/ml control peptide for 1 h at 4°C prior to the induction with 20 ng/ml biotinylated human HGF, 20 ng/ml biotinylated human VEGF₁₆₅ (as performed for HGF) or 20 ng/ml biotinylated soybean trypsin inhibitor (used as negative control) (R&D Systems) for 1 h on ice. The cells were then incubated with an avidin–HRP conjugate (Abcam) for 30 min at 4°C protected from light. Cells were washed twice with PBS and the ABTS solution (Roche Diagnostics) was added for 45 min at room temperature. The absorption at 415 nm was determined with an ELISA reader EXL 808 (Biotek). Experimental data of the negative controls (biotinylated soybean trypsin inhibitor) were directly subtracted from the experimental data of the biotinylated human HGF and VEGF₁₆₅.

96-well-plates were blocked overnight with PBS supplemented with 4% BSA. The following day, 1 x 10⁶ cells per well were blocked for 30 min on ice with PBS supplemented with 4% BSA. 100-150 µl bacterial supernatant containing approximately 1 x 10⁹ phages were mixed with the cells and incubated for 1 h at 4°C. CD7-targeting scFv phage was used as control (32 ). Plates were washed 3-times with cold PBS supplemented with 0.1% BSA and subsequently incubated for 1 h at 4°C with anti-M13-HRP antibody (Creative Diagnostics; cat. no. CAB-655M; diluted 1:2000 in PBS supplemented with 4% BSA). After washing as described above 100 µl ABTS solution/well (Roche; cat. no. 11112422001) was added. Absorbance at 405 nm was measured with Sunrise absorbance microplate reader (Tecan; reference wavelength 492 nm).