Titrations of 10 mM quinate or shikimate dissolved in the same buffer were performed with either buffer or 200 μM wild-type or K170M DHQD.
Heat induced antigen retrieval in pH 9.0 EDTA buffer was performed and samples were blocked with peroxide blocking solution from DAKO DAKOO, Hamburg, Germany).
For C. albipunctata embryos, the following modifications to the staining protocol apply: proteinase K treatment was carried out for 7 min at room temperature; post-hybridization washes: an additional wash of 10 min 2xSSC/hybridization buffer was performed before washing for 15 min with PBT/hybridization buffer; antibody incubation: embryos were incubated with anti-DIG for 2 hrs.
A subsequent dilution with additional buffer was performed to lower the protein concentration to 8 μM.
The eluted protein was concentrated, and a buffer exchange was performed with 25 mM phosphate buffer containing 10 µM FAD.
One mL of each sample was desalted through a 10 DG column (Bio-Rad, USA) and buffer exchange was performed with 10 mM Tris.Cl, pH 6.8.
Paraffin-embedded formalin-fixed 3 μm sections were deparaffinized with EZPrep buffer, antigen retrieval was performed with CC1 buffer (both Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex, Richmond, CA).
After two washes with PBS-0,05% tween buffer followed with two washes with a pH 7,5 phosphate-citrate-0,05% tween buffer (CT), revelation was performed with 200 µl of a freshly solution of ortho-penylene diamine (OPD; Sigma Aldrich) at 1,5 mg/ml in CT buffer containing 0,075% hydrogen peroxide.
After boiling in SDS buffer, western blotting was performed with specific antibodies to visualize proteins interacting with α4.
After 3 washes with 0.05% Tween-20 in PBS (washing buffer), a second incubation was performed with 100 μL of biotin-conjugated anti-human IgG (Sigma, St Louis, Mo, USA), diluted 1 : 2000 in saturation buffer, at 37°C for 1 h.
