Mouse monoclonal antibody against beta actin

This experiment was performed in order to assess the baseline expression of SR‐BI, the receptor for our HDL‐mimicking nanoparticles (HPPS), in different cancer cell lines. The expression of SR‐BI on the cell surface is a requirement for uptake of HPPS. KB, HT1080, Hep3B, SNU398, and Huh7 HCC cell lines were seeded on a six‐well plate at a density of 2 × 10⁵ cells per well and grown for 24 hours. Cell lysates were prepared with radio immunoprecipitation assay buffer plus complete protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL) and immunoblotted using antibodies specific for SCARB1 (anti‐SR‐BI antibody EP1556Y, 1:1,000; Abcam, Inc.). Immunoreactive proteins were detected using goat anti‐mouse horseradish peroxidase‐conjugated secondary antibody (GenScript, Pascataway, NJ) and Clarity Western enhanced chemiluminescence (Bio‐Rad Laboratories Ltd., Ontario, Canada). Membranes were stripped and immunoblotted with a mouse monoclonal antibody against beta‐actin (1:5,000; Sigma). Imaging was carried out using a Gel Logic 2200 Imaging System (Kodak, Rochester, NY).

The miRNeasy isolation kit and the QuantiNova SYBR Green PCR mix were purchased from Qiagen (Valencia, CA). Running buffer, transfer buffer, NuPage 4–12% Bis-Tris gels, and Invitrolon polyvinylidene fluoride (PVDF) membranes (0.45 µm) were procured from Invitrogen (Carlsbad, CA) while the blocking buffer was from Li-Cor Biosciences (Lincoln, NE). Antigen retrieval solution and 4',6-diamidino-2-phenylindole (DAPI) were from Vector Laboratories (Burlingame, CA), and the normal donkey serum was purchased from Jackson ImmunoResearch Laboratories (Westgrove, PA). The following primary antibodies were used for immunohistochemistry and western blotting: rabbit monoclonal antibody against aquaporin 5 (1:100 or 1:5,000; Abcam Inc., Cambridge, MA); mouse monoclonal antibody against E-cadherin (1:100 or 1:5,000; BD Biosciences, San Jose, CA), rabbit polyclonal antibody against α-SMA (1:100 or 1:5,000; Abcam Inc.), and mouse monoclonal antibody against beta actin (1:10,000; Sigma-Aldrich Co., St. Louis, MO). Fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC) conjugated secondary antibodies (1:100, Jackson ImmunoResearch Laboratories) were used for immunohistochemical staining, while IRDye 680RD or IRDye 800CW secondary antibodies (1:5,000; Li-Cor Biosciences) were used for western blotting.