Fig 1: LDL binding to cell receptors measured by ELISA at pH 7.4. A–C: Validation assays for binding of control LDL to cell receptors; (A) LDLR, (B) CD36; (C) LOX-1. The receptors were immobilized on the wells and LDL binding was measured by ELISA using increasing concentration of control LDL that were either intact (for binding to LDLR) or have been oxidized by copper as described in Materials and methods (for binding to CD36 and LOX-1). The absorbance values were corrected for nonspecific binding using BSA-coated wells as a control. The binding was measured using increasing LDL concentrations at pH 7.4. D–F: Measurements of LDL binding to cell receptors; (D) LDLR, (E) CD36, (F) LOX-1. LDL protein concentration was 0.5 μg for LDLR, 5 μg for CD36, or 10 μg for LOX-1. Each data point represents an average of five independent measurements. The bars show median ±SD; ns, not significant, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. LDLR, LDL receptor.
Fig 2: Binding of soluble Siglec-5 and Siglec-14 to E. coli-NadA. Binding of rh-Siglec-5, rh-Siglec-14, rh-Siglec-9, and rh-LOX-1 to E. coli-NadA was revealed by flow cytometry (A and B) and confocal microscopy (C). E. coli-NadA (A and B, top panels) and E. coli-pET (A and B, bottom panels) bacteria were incubated with 1µg/mL of Siglec-5, Siglec-14, Siglec-9, and rh-LOX-1 prior to the incubation with polyclonal anti-NadA sera. Siglec-NadA complexes were revealed by 488-anti-human and 568-anti-mouse secondary antibodies, while Siglec-LOX interaction was revealed with 488-anti-rabbit and 568-anti-mouse secondary antibodies. Bacteria incubated with secondary antibodies alone were used as negative controls, while bacteria stained exclusively with anti-NadA sera were used to verify the expression of NadA. (C) For confocal microscopy E. coli-NadA cells were stained as for FACS analysis and then immobilized on polylysine-coated glass slide. DAPI was used to stain bacterial chromosome (blue). Scale bars represent 2 µm.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human LOX-1/OLR1 Protein, CF