Journal: PLoS ONE

Article Title: A Subregion of Reelin Suppresses Lipoprotein-Induced Cholesterol Accumulation in Macrophages

doi: 10.1371/journal.pone.0136895

Figure Lengend Snippet: (A) ELISA plates were coated with c-myc peptide, purified R5-6 or R5-6C, or null eluent from empty-vector transformed bacteria as a control (Ctrl), and then incubated with RAW 264.7 cell lysates. The apoER2 and VLDLR in RAW 264.7 cell lysates bound to ELISA plates were detected using antibodies against apoER2 and VLDLR. (B) RAW 264.7 cells were transfected with empty pcDNA3.1B vector or apoER2-GFP expression plasmid, and then incubated with 30 μg/ml of apoB-carrying and apoE-free lipoproteins (+LP) or without lipoproteins (-LP) in the presence or absence of 0.2 μg/ml purified R5-6 protein. The amount of R5-6 bound to the cell surface was determined by ELISA using an antibody against c-myc epitope. The background binding was determined using a normal mouse IgG. (C-D) RAW 264.7 cells were treated with 0.2 μg/ml of purified R5-6 protein or culture medium alone (Ctrl). The protein level of ABCA1 was determined by immunoblotting and quantified relative to β-actin. (E) RAW 246.7 cells were treated as described in plane B. The mRNA level of ABCA1 was determined by quantitative real-time RT-PCR and normalized relative to GAPDH mRNA. (F) RAW 264.7 cells were labeled with 0.25 μCi/ml of 3 H-cholesterol, followed by incubation with 5 μg/ml c-myc peptide, 0.2 μg/ml purified R5-6 protein or vehicle control (Ctrl). Cholesterol efflux was determined in cells incubated with or without apoAI treatment, and was expressed as the percentage of radioactivity in the medium compared to the total radioactivity in the cells and medium. ApoAI-mediated cholesterol efflux was calculated as the difference of efflux from cells in the presence and absence of apoAI treatment. Data represent the mean ± SEM of four or more independent experiments. * p < 0.05 vs . Ctrl; † , p <0.05 vs . c-myc-immobilized ELISA plates or treated cells; ‡ , p < 0.05 vs . cells transfected with the same plasmids and untreated with R5-6; $ , p < 0.05 vs . cells transfected with empty vector and treated with R5-6; and # , p < 0.05 vs . cells transfected with apoER2-GFP-expression plasmid and untreated with lipoproteins.

Article Snippet: PI3K inhibitor LY294002 (sc-201426), Sp1 inhibitor mithramycin A (sc-200909), ELISA plate (sc-204463), scrambled siRNA, and siRNAs specific for VLDLR or apoER2, antibodies against ABCA1 (sc-58219), β-actin (sc-47778), apoER2 (sc-20746), VLDLR (sc-18824), and mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Plasmid Preparation, Transformation Assay, Bacteria, Control, Incubation, Transfection, Expressing, Binding Assay, Western Blot, Quantitative RT-PCR, Labeling, Radioactivity