Anti abca1 antibody

Cells were lysed with lysis buffer [1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 mM Tris-HCl, pH 7.5, phosphatase inhibitor cocktail I and II (Sigma-Aldrich), 50 mM dithiothreitol, 150 mM NaCl, and complete protease inhibitor cocktail (Roche Diagnostics)], after which cell lysates were resuspended in 5×Tris-glycine SDS sample buffer. The protein concentration was assayed using Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein from each sample were subjected to SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Pall, Glen Cove, NY, USA). The immunoblots were blocked with 5% non-fat milk in PBST (5.8 mM Na₂HPO₄·7H₂O, 130 mM NaCl, and 0.05% Tween-20) at room temperature and incubated with anti-ABCA1 antibodies (Abcam, Cambridge, UK) overnight at 4°C. After the blots were washed with PBST, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich), and target protein bands were detected using an enhanced chemiluminescence system (ECL; PerkinElmer). Next, the blots were stripped to allow further probing with anti-α-tubulin antibodies as an internal control for the amount of total cellular protein. The relative intensities of the protein bands were quantified by densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

Cells and tissues were harvested and protein extracts prepared using established methods. Extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subjected to western blot using rabbit polyclonal anti-ABCA1 antibodies (Abcam Inc., Cambridge, MA, USA) and rabbit polyclonal anti-β-actin antibody (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA). Proteins were visualized using the chemiluminescence method (ECL Plus Western Blot Detection System; Amersham Biosciences, Foster City, CA, USA).

BMDMs were seeded onto 6 well plates. After treatment as described above, cells were washed with PBS and lysed in HEPES buffer (20 mM HEPES PH7.2, 50 mM NaCl, 0.5% TritonX-100, 1 mM NaF, 1 mM DTT, 5 mM EDTA) containing protease inhibitors on ice. The lysates were collected and boiled for 20 min and separated on 10% SDS-PAGE, then transferred to nitrocellulose membranes, blocked with skim milk, incubated with primary antibodies and specific secondary antibodies, and specific proteins were detected using chemiluminescence method and the bands intensities were scanned and calculated by densitometry (NIH ImageJ software). Relative amounts of each protein were normalized by calculated. These following antibodies were used. Anti-SR-B, ABCG1 and β-actin antibodies were purchased from Santa cruz. Anti-CD36, SR-A, LOX-1 antibodies were purchased from R&D system, and anti-ABCA1 antibody was obtained from Abcam. Anti-p-p38, p-p65, p-IκB, p-erk, p-JNK antibodies were from Cell Signaling Technology.