Fig 1: Immunoreactivity of heated and glycated BLG after enzymatic digestion in infant gastrointestinal static in vitro model. BLG: Non‐treated, heated (BLG‐H), heated with lactose (BLG‐Lac) or heated with glucose (BLG‐Glu) were subjected to gastric and intestinal phase of enzymatic digestion and immunogenicity of digested samples was measured as (A) binding to sRAGE, Gal‐3, CD36 and SR‐AI in inhibition ELISAs; following positive controls were used: amyloid‐β for sRAGE and CD36, fucoidan for SRAI and soy protein extract glycated with glucose for Gal‐3. B) binding assay to THP‐1 macrophages using Alexa Fluor 647 conjugated anti‐BLG antibodies for flow cytometry detection. Digestion blank (dBlank) – sample containing all digestive enzymes but no BLG. Data shown as mean ± SD of triplicate wells and are representative of at three (A) or two (B) independent experiments. Significant differences analyzed with one‐way ANOVA with Tukey post hoc comparison test (GraphPad Prism); *p < 0.05; **p < 0.01, ***p < 0.001.
Fig 2: Inhibition of binding and internalization of processed BLG by human THP‐1 macrophages. PMA differentiated THP‐1 macrophages were pre‐incubated with endocytosis inhibitors: dynasore and cytochalasin D (Cyt D) or inhibitors of specific receptors: amyloid‐β and FPS‐ZM1 for RAGE; Oxidized low‐density lipoprotein (Ox‐LDL) for CD36; fucoidan for SR‐AI, short peptides 33‐DFTG and G3‐C12 for Gal‐3. Followed by pre‐incubation with inhibitor the cells were incubated in a presence or absence of BLG: non‐treated (BLG‐NT), heated (BLG‐H), heated with lactose (BLG‐Lac) or heated with glucose (BLG‐Glu) and stained extracellularly (A) or intracellularly (B) with Alexa Fluor 647 conjugated anti‐BLG antibodies. The level of fluorescence was measured by flow cytometry and expressed as a relative to non‐inhibited THP‐1 cells. Data shown as mean ± SD of triplicate wells and are representative of three independent experiments. Significant differences analyzed with one‐way ANOVA with Tukey post hoc comparison test (GraphPad Prism); *p < 0.05; **p < 0.01, ***p < 0.001.
Fig 3: sRAGE dot blot volume intensities (top) and dot blot images (bottom). Cow’s milk protein (MP). MP was non-treated (NT-MP), heated at low temperature (LT-MP), and heated at high temperature (HT-MP), while soy protein, glycated with glucose for 90 min at 100 °C (G90), was used as positive control.
Fig 4: Overview of the experimental design. Non-treated milk protein (NT-MP), low-temperature-heated milk protein (LT-MP), high-temperature-heated milk protein (HT-MP), casein depleted (CN-DP), gastric phase (GP), intestinal phase (IP), water activity (aw), and soluble receptor for advanced glycation end products (sRAGE).
Fig 5: (a) sRAGE inhibition ELISA on 200 µg/mL cow’s milk protein (MP), non-treated (NT-MP), low-temperature-heated (LT-MP), high-temperature-heated (HT-MP), and NT-MP after acidic depletion of casein (CN-D). (b) sRAGE inhibition ELISA and (c) CD36 inhibition ELISA on micellar casein (CM) as well as sodium caseinate (CN) measured at different concentrations. Amyloid-β (AMB) and soy protein glycated with glucose for 90 min at 100 °C (G90) were used as positive controls, while ovalbumin (OVA) was used as a negative control. Statistical differences in (a) were determined using one-way ANOVA. Values were considered as statistically different at p < 0.05, with different letters indicating statistically significant differences between samples.
Supplier Page from BioVendor Laboratory Medicine, Inc. for Soluble Receptor for Advanced Glycation End Products Human E. coli