Abca1

The expression of proteins directly involved in lipogenesis (Fas; Cell Signaling, Beverly, MA, USA), oxidation pathway (Cpt1, β-had; Santa Cruz Biotechnology, Dallas, TX, USA) and the process of desaturation and elongation (Elovl3, Elovl6, Scd1; Santa Cruz Biotechnology) as well as fatty acid exporting proteins: Abca1 (Thermo Scientific, Waltham, MA, USA) and Mtp (Santa Cruz Biotechnology) were detected by routine Western Blotting as previously described in details by Konstantynowicz-Nowicka et al. [28 (link)]. Briefly, protein concentration was determined using bicinchonic acid method (BCA) with bovine serum albumin (BSA) as a standard. Signals obtained by immunoblotting were quantified densitometrically using a ChemiDoc visualization system (Bio Rad, Warsaw, Poland). Equal protein loading was confirmed using Ponceau S staining. The expression of all the proteins was standardized to the Gapdh (Santa Cruz Biotechnology) expression and the control was set at 100%.

Routine Western Blotting procedures were used to detect the total expression of fatty acid transport proteins: FATP2, FATP5 (Santa Cruz Biotechnology, USA), FAT/CD36, FABPpm (Abcam, UK), ABCA1 (Thermo Scientific, USA), MTP (Santa Cruz Biotechnology, USA), ACBP and L-FABP (Abcam, UK) as well as the proteins directly involved in lipogenesis (FAS; Cell Signaling, USA), oxidation pathway (CPT 1; Santa Cruz Biotechnology, USA) and lipid metabolism (PPARα, LXR, SREBP1c, pAMPK; Cell Signaling, USA) as previously described in details by Konstantynowicz-Nowicka et al. [17 ]. All the antibodies used in our procedures were monoclonal except for FATP2 and FAT/CD36. Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk, the membranes were immunoblotted with primary antibodies of interest and incubated with secondary antibodies labeled with horseradish peroxidase (HRP). Obtained protein bands were quantified densitometrically using a ChemiDoc visualization system (Bio Rad, Warsaw, Poland). Equal protein loading was controlled by Ponceau S staining. The expression of all the proteins was standardized to the GAPDH (Santa Cruz Biotechnology, USA) expression and the control was set as 100%.

Peritoneal macrophages were obtained by injecting 3 mL of 4% Brewer’s thioglycollate (Merck) intraperitoneally followed by a collection of the elicited peritoneal exudate cells for 4 days after injection. Exudate cells were centrifuged to use as ex vivo samples or resuspended in RPMI medium (Life Technologies, Carlsbad, CA, USA) with 100 U/mL of penicillin and 100 μg/mL of streptomycin (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum (Lonza, Walkersville, MD, USA) at 37 °C with 5% CO₂. The cells were then incubated with or without 40 μg/mL of ox-LDL (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 24 h. In KD examination, AT1R, ELAVL1, PPARγ, ABCA1, and control siRNA (Thermo Fisher Scientific) were transfected with lipofectamine (Thermo Fisher Scientific) and the mRNAs of interest were analyzed by real-time PCR after 24 h. For the scratch assay, cells were grown to confluence on 24-well tissue dishes, and a single scratch was made using a sterile 1000-μl pipette tip. Photographs were taken after 12 h, and the cell-covered area was measured by Image J. In overexpression examination, ABCA1 cDNA clones (Santa Cruz Biotechnology Inc.) were transfected with UltraCruz Transfection Reagent (Santa Cruz Biotechnology Inc.) for 24 h, and then ox-LDL were added. The mRNAs of interest were analyzed by real-time PCR after 24 h.