Cumaric acid

3,4,5-trihydroxybenzoic acid (GA), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,4-dihydroxybenzoic acid (PCA), 5-caffeoylquinic acid (5CQA), 2,5-dihydroxybenzoic acid (2,5-DHBA), catechin (Cat), 4-caffeoylquinic acid (4CQA), p-hydroxybenzoic acid (pHBA), vanillic acid (VA), caffeic acid (CA), syringic acid (SyA), m-hydroxybenzoic acid (mHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), cumaric acid (CuA), sinapic acid (SA), ferulic acid (FA), rutin (Ru), myricitrin (My), quercetin-3-O-glucoside (Q3G), kampferol-3-O-rutinoside (K3R), salicylic acid (SaA), hesperidin (He), quercetin (Q), kampferol (K), isoxanthohumol (IsoX), xanthohumol (X), formic acid, and dichloromethane, were purchased from Sigma-Aldrich (Milano, Italy). HPLC-grade acetonitrile and methanol were Carlo Erba (Milano, Italy); HPLC-grade water was prepared with the Milli-Q purification system (Millipore, Vimodrone, Italy).

Sodium Dodecyl Sulfate–Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed following standard methods [67 ] using the Miniprotean III system (Bio-Rad). The sample buffer for reducing SDS-PAGE is 60 mM Tris–HCl pH 6.8, 1% w/v SDS, 5% v/v glycerol, 0.005% w/v bromophenol blue and 1% v/v 2-mercapto-ethanol (2-ME). In non-reducing SDS-PAGE, the sample buffer was devoid of 2-ME. Proteins separated by SDS-PAGE were either stained with Coomassie Blue R-250 (Bio-Rad) or subjected to Western blot. For the latter, the proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Immobilon-P, Millipore) using semi-dry electrophoresis (Bio-Rad), as previously described [67 ]. Nb-HlyA fusions were detected with primary mAb anti-E-tag (Phadia, 1:5000) and secondary polyclonal rabbit anti-mouse IgG antibodies fused to POD (1:5000, Sigma). Membranes were developed using a mixture of 100 mM Tris–HCl (pH 8.0) containing 1.25 mM luminol (Sigma), 0.22 mM cumaric acid (Sigma), and 0.0075% (v/v) H₂O₂ (Sigma). The membranes were then developed by exposure to X-ray films (AGFA).