Dii ox ldl

The collected bone marrow-derived macrophages as above were resuspended in the conditioned media at 5.0 × 10⁶ cells/mL. The 100 μL cell suspension (5.0 × 10⁵ cells) was placed into each well of a 96-well plate, and the cells were incubated at 37 °C in a 5% CO₂ incubator for 16 h to settle. The media were removed, and the cells were washed twice with warm HBSS. The cells were incubated with 100 μL RPMI1640 containing DiI-oxLDL (20 µg/mL; Biomedical Technologies) at 37 °C in a 5% CO₂ incubator for 5 h. The DiI was subsequently extracted from the cells using 100 μL of 2-propanol. The fluorescent intensity was measured using an ABI prism 7700 (Applied Biosystems, Foster City, CA, USA) using the Joe channel (absorption at 520 nm and emission at 548 nm). Various concentrations of murine wild-type (wt) Pg and an active site mutant [S⁷⁴³A]-Pg recombinantly synthesized by S2 cells, which are derived from Drosophila melanogaster, without bovine serum [25 (link)], along with aprotinin (USB Corporation, Cleveland, OH, USA), were added to the culture to evaluate the functions of Pg and Pm. We have chosen Drosophila S2 cells without fetal bovine serum, as opposed to mammalian cell lines, to express recombinant Pg because of the ubiquitous presence of hPg activators in mammalian cells.

DMEM was purchased from Gibco (Life Technologies). Human copper‐oxidized low‐density lipoprotein (ox‐LDL) and DiI‐Ox‐LDL were obtained from Biomedical Technologies. NPY (ab120208), Y1, Y2 and Y5 antagonists were obtained from Sigma. HDL (L8039) and apolipoprotein A‐I (ApoA‐I, SRP4693) were obtained from Sigma.
Anti‐PKC (SAB4502354), Anti‐p‐PKC (SAB4301254), anti‐JAK2 (SAB4501601) and anti‐p‐JAK‐2 (SAB4300124) antibodies were purchased from Sigma. AntiABCA1 (NB400‐105) and antiABCG1 (NB400‐132) antibodies were purchased from Novus Biologicals, Inc. PPARγ, anti‐CD36 (sc‐9154), anti‐SRA and anti‐β‐actin were obtained from Santa Cruz Biotechnology, Inc.