Mab 10005

Antibody against human ABCA1 (MAB 10005) was purchased from Merck. Antibodies against pFAK³⁹⁷ for immunostaining (#44-625G), anti-rabbit IgG HRP (#31466), anti-mouse IgG HRP (#62-6520) anti-rabbit IgG—Alexa Fluor 488 (#A-21206), anti-mouse IgG—Alexa Fluor 647 (#A-3157) were purchased from Invitrogen. Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK³⁹⁷ for Western blot (D20B1) from Santa Cruz. Antibodies against pCREB¹³³ (#9191) and pSTAT3⁷²⁷ (#9134) were purchased from Cell Signaling Technology. Proteinase and phosphatase inhibitors were purchased from Thermo Fischer Scientific, bovine serum albumin (BSA) and dimethyl sulfoxide (DMSO) from Bioshop, probucol and gelatin from pork skin were from Sigma-Aldrich, cholesterol was from Northern Lipids Inc. and methyl-β-cyclodextrin (MβCD) was from Alfa Aesar. All commonly used biochemical reagents stated below were purchased either from Sigma-Aldrich or Bioshop.

A total of 0.5 μl of mouse plasma or 20 μg of Panc02 RIPA total protein extracts were analyzed by reducing SDS–PAGE (8% PAA gel) and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with TBS (20 mM Tris–HCl, pH 7.4, 137 mM NaCl) containing 5% (wt/vol) fatty acid–free BSA or nonfat dry milk and 0.1% Tween-20 (blocking buffer) for 1 h at room temperature. Proteins of interest were detected with antibodies directed against APOA1 (in-house produced rabbit polyclonal antihuman APOA1 antibody), ABCA1 (MAB10005; Merck), β-actin (clone AC-74; Sigma-Aldrich), phospho-AKT (S437, 4060S; Cell Signaling), phospho-AKT (Thr308, 13038S; Cell Signaling), and pan AKT (2920S; Cell Signaling) followed by incubation with HRP-conjugated secondary antibodies and development with the enhanced chemiluminescence protocol (Pierce).