Figure 5
Biotinylated LDL and VLDL bind CLCF1. (A) Interaction between CLCF1 and lipoproteins was assessed using ligand blotting assays: 0.1–0.2 μg of hCLC, or 1 μg of BSA were subjected to non-reducing SDS-PAGE. Proteins were revealed by Coomassie blue staining (left section) or electro-transferred to nitrocellulose and incubated successively with 30 μg/ml biotinylated VLDL, LDL or HDL and HRP-labeled neutravidin (middle 3 panels). Signal was revealed by chemiluminescence. Image in the far right section shows results of incubation with HRP-labeled neutravidin alone. Lane hCLC biot; 40 ng of biotinylated CLC was used as positive control for neutravidin detection. Blots’ images where cropped to show relevant areas. (B) The formation of LDL and VLDL complexes can be detected using an AlphaLISA proximity assay. Biotinylated hCLCF1 was mixed with LDL, HDL and VLDL covalently coupled to acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.
