Caveolin 1 antibody

Protein levels of enriched caveolae were determined by the Bradford protein assay (BioRad, Cat # 5000001). Quantities of 1, 2 and 5 µg of protein were ran through 15% acrylamide gel with a 5% stacking gel for cav-1, ER-α and Her-2/neu, respectively, to determine protein expression by Western protein blotting. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, blocked overnight in 5% skim milk powder and incubated for 2 h at room temperature with either caveolin-1 antibody (Santa Cruz, Cat # sc-894) diluted 1:500 or ER-α antibody (Santa Cruz, Cat # sc-7207) diluted 1:200 or Her2 neu antibody (Santa Cruz, Cat # sc-101695) diluted 1:200. All primary antibodies were followed by goat anti-rabbit IgG HRP (Santa Cruz, Cat # sc-2030) diluted 1:1000 for 1 h at RT. Protein bands were detected by Western Lightening Plus ECL (Perkin Elmer, Cat # NEL 103001EA) and visualized on a FluorChem HD2 imager (Cell Biosciences, Santa Clara, California, USA). Bands were quantified using AlphaView imaging software (Version 3.1.1.0).

Cells were scraped with lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) on an ice tray, and cell lysates were subjected to western blot analysis. Protein samples were first separated by SDS–PAGE and then transferred to a PVDF membrane. Primary antibodies were applied to detect specific protein expressions, followed by incubation with appropriate HRP-conjugated secondary antibodies. Protein signals were developed using an enhanced chemiluminescence reagent (Millipore/Merck, Billerica, MA, USA) and detected by Multigel-21 digital system (Hung Chong, Taiwan) that revealed the relative intensity and compared to control group in the experiment. Western blots were carried out with caveolin-1 antibody (Santa Cruz, Dallas, TX, USA) and a marker for apoptosis was characterized using Bcl-2 antibody (Genetex, Irvine, CA, USA); markers for cell survival such as phosphor-mTOR, AKT, and MAPK antibodies (Cell Signaling Technology, Danvers, MA, USA) were also identified. Actin and GAPDH antibodies were used as loading control depending on the molecular weight of detecting proteins.