Supplementary legends to the figures
Figure S1: Analysis of on-site prepared cashew protein extracts by SDS PAGE. Protein fraction was extracted from defatted cashew flour as indicated in material and methods. This extract was denatured and analyzed by SDS PAGE (lane 2) in comparison to initial cashew flour (lane 1) and commercial cashew protein extract (Stallergenes Greer, lane 3). The relative molecular mass (kDa) is indicated on the left. The position of major allergens are indicated on the right, based on previous published data . Note that Ana o 2 separates as two distinct bands following denaturation since it is constituted by two subunits (acidic and basic) linked by disulfide bonds.
Figure S2: Analysis of cashew protein extracts recovered from patches. Epicutaneous patches were loaded with commercial cashew protein extracts (Stallergenes Greer) in liquid form, by dissolving protein lyophilizate in PBS 1X, NaCl 0.45% or phosphate buffer 0.1 M. Patches were dried for one hour at 30°C. Then, proteins were redissolved from patch backing using distilled water. (A ) Recovered proteins were used as a coating in ELISA to analyze their capacity to bind to cashew-specific antibodies induced in mice by oral sensitization. HRP-conjugated anti-mouse IgG1 (left panel) or anti-mouse IgE (right panel) were used as secondary antibodies. Optical densities (O.D.) at 450 nm were plotted against plasma dilution, (n = 7 per group). Data are median and range of individual values and non-linear regression curve was obtained using sigmoidal 4PL equation. (B ) Recovered protein extracts were centrifuged and total extract (before centrifugation, T), supernatant (S) and pellet (P) were denatured and analyzed by SDS PAGE. The relative molecular mass (kDa) is indicated on the left.