Anti sr b1 antibody

Liver homogenates (40 μg) were separated by SDS PAGE on 7.5% polyacrylamide gels under reducing conditions and subject to western blot analysis using an anti-low density lipoprotein receptor (LDLR) antibody (Abcam, Cambridge, ab30532), an anti-HMGCoA reductase antibody (Abcam, ab174830), an anti-SR-B1 antibody (Novus Biologicals, Littleton, CO, NB400-113) and an anti-actin antibody (Sigma, St Louis, MI). Blots were washed and then incubated with a goat anti-rabbit IgG-hrp antibody (Thermo Scientific, Waltham, MA). Membranes were developed using enhanced chemiluminescence (ECL) on the LI-COR Odyssey (LI-COR Biosciences Inc, Lincoln, NE). Protein quantification was performed by Image Studio Lite (LI-COR Biosciences, Inc) with protein normalised against actin.

The SR-B1 receptor was blocked with 5 μg/ml of anti-SR-B1 antibody (Novus Biological, Littleton, CO) preincubated with HepG2 cells for 3 h at 37°C prior to Lp(a) treatment. The SR-B1 receptor was also blocked with an inhibitor, Block Lipid Transport-1, BLT-1 (Merck Millipore, Darmstadt, Germany), preincubated with HepG2 cells at 100 μM for 1 h at 37°C prior to Lp(a) treatment. Apo(a) internalization was blocked with an anti-apo(a) polyclonal antibody (Wako, Richmond, Virginia) preincubated with 5 μg/ml Lp(a) for 1 h at room temperature prior to incubation with HepG2 cells. In addition to blocking with anti-apo(a), the oxPLs on Lp(a) were blocked with 5 μg/ml of the E06 antibody (Avanti Polar Lipids, Alabaster, AL) prior to Lp(a) treatment. RNA and protein were extracted from treated cells and analyzed for the ABCA1, PPARγ, and LXRα mRNA and protein levels, as described above.

Peripheral blood and bone marrow cells were isolated from mice treated as described. Single-cell suspensions from the aorta were also prepared according to the protocol described before64 (link). Cells were stained with anti-Ly6C, anti-Ly6G, anti-CD11b and anti-CCR5 antibodies (BioLegend), and anti-SR-B1 antibody (Novus). The samples were then analysed by FACSCanto II (BD Biosciences) and monocytes were gated within Ly6G-negative population. The data were processed by FACSDiva (BD Biosciences) or Flow Jo (Tree Star).