Anti abca1

IU1 (S7134), obtained from Selleckchem (Houston, TX, USA), was dissolved into DMSO and stored at −20°C. USP14 siRNA (sc‐76817) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐GAPDH (MB001) was purchased from Bioworld Technology (St. Louis Park, MN, USA). Anti‐CD36 (ab133625), anti‐ABCG1 (ab52617), anti‐SR‐B1 (ab217318) and anti‐SR‐A (ab123946) were from Abcam. Anti‐Ubiquitin and anti‐USP14 were obtained from Cell Signaling Technology (MA, USA). Anti‐ABCA1 was from Santa Cruz, and Anti‐Lox‐1 was from R&D system. Oil Red O was purchased from Sigma‐Aldrich. Human oxidized low‐density lipoprotein (oxLDL) (YB‐002) and human Dil‐labelled oxidized low‐density lipoprotein (Dil‐oxLDL) (YB‐0010) were purchased from Yiyuan Biotechnologies (Guangzhou, China). Dynabeads antibody coupling kit was obtained from Life technologies. PMA (P1585) was from Sigma‐Aldrich. Anti‐CD36 (FITC) (ab39022) was purchased from Abcam.

Total protein was extracted and the levels were determined using a BCA Protein Assay Kit. Equal amounts of total protein (50 μg) were loaded and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were then blocked in 5% BSA for 1.5 h, followed by overnight incubation at 4 °C with the following primary antibodies: anti-β-actin (1: 1000 dilution), anti-ABCA1 (1: 1000 dilution), anti-Nrf2 (1: 1000 dilution), anti-HO1 (1: 1000 dilution), and anti-LOX1 (1: 1000 dilution) (purchased from Santa Cruz Inc., California, USA). Anti-p38 MAPK (1: 1000 dilution), anti-p-p38 MAPK (1: 1000 dilution), anti-ERK1/2 (1: 1000 dilution), anti-p-ERK1/2 (1: 1000 dilution), anti-JNK (1: 1000 dilution), anti-p-JNK (1: 1000 dilution), anti-Akt (1: 1000 dilution), and anti-p-Akt (1: 1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies, horseradish peroxidase (HRP)-labeled goat-anti-rabbit and goat-anti-mouse IgG, were purchased from Wuhan Boster Bio-engineering Co. Ltd. (Wuhan, China). Blots were processed for enhanced chemifluorescence using a Pierce ECL western blotting substrate (Thermo Scientific Pierce, Rockford, IL, USA).

Caco-2 cells were treated with or without 1 mM FP for 24 h. After treatment, cells were washed twice with ice-cold phosphate-buffered saline and lysed with 100 μL of NUN buffer containing 0.33 M NaCl, 1.1 M urea, 1% Nonidet P-40, 25 mM HEPES (pH 7.6), and proteinase inhibitors (Roche, Basel, Switzerland) by direct addition to the plate. Cell lysates were collected and cleared by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants were collected as whole-cell lysates. The cell lysates were separated by 8% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). After blocking, the membranes were incubated with the following specific antibodies: anti-ABCA1 (sc-58219, Santa Cruz Biotechnology), anti-NPC1L1 (sc-166802, Santa Cruz Biotechnology), anti-LXRα (NBP1–77106, Novus Biologicals), anti-LXRβ (NBP100–74457, Novus Biologicals), and anti-β-actin (sc-47778, Santa Cruz). Immunoblot analyses were performed using an ImmunoStar^® LD system (Wako Pure Chemical Industries).