Western lightening plus ecl

Several washes with TBS-Tween (150 mM NaCl, 10 mM Tris, 0.05% Tween20) were performed. The membrane was blocked with 5% milk in TBS-Tween for 1 h. Overnight incubation at 4 °C using primary antibodies was carried out. The primary antibody was CT-CX46, with an antigen sequence: CRLPSRNSRHSSNRS (1:250, GeneScript, Piscataway, NJ, USA). The secondary antibody incubation was performed for 1 h using anti-rabbit peroxidase-conjugated antibodies (1:1000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The membrane was washed with TBS-Tween for 10 min three times. Finally, the membrane was revealed with a chemiluminescent solution (Western Lightening Plus-ECL, Perkin Elmer, Waltham, MA, USA) in the chemiluminescent and fluorescent equipment (Odyssey FC, Li-COR, Lincoln, NE, USA). For dot blot experiments, 25 μg of total protein extract was used and a similar detention procedure was used.
Densitometric quantification was achieved using Image J software (NIH). The signal associated with cx46 which included the signal area (ROI) and the intensity was performed using the parametric software’s tool plugin. The measures corresponded to the dynamic range over the background signal. Only a clear and distinguishable peak from the background signal was used. Values below 110 arbitrary units (background) were not considered.

Total cell lysates from human RMS cell lines were obtained following lysis in 2%SDS lysis buffer supplemented with protease inhibitors (Santa Cruz Biotechnology, Dallas, Texas). Samples were boiled, vortexed and homogenized through a 28G syringe. 20–40 μg of protein was loaded in 4–20% Mini-Protean TGX gels (Biorad, Hercules, CA) and transferred onto PVDF membranes. Western blot analysis used primary antibodies: rabbit a-MYF5 (1:5000, Abcam ab125078, Cambridge, MA), mouse a-MYOD1 (1:1000, Abcam ab16148, Danvers, MA), rabbit a-MYOD1 (1:1000, Abcam ab133627), rabbit a-GAPDH (1:2000, Cell Signaling 2118), mouse a-TUBULIN (1:2500, Abcam ab4074) and secondary antibodies: HRP anti-rabbit (1:2000, Cell Signaling 7074) or HRP anti-mouse (1:3000, GE Healthcare NA93IV, Marlborough, MA). Blocking was completed using 5% skim milk/TBST. Membranes were developed using an ECL reagent (Western Lightening Plus-ECL, Perkin Elmer, Waltham, MA or sensitive SuperSignal West phemto Maximum Sensitivity Substrate, Thermo Scientific, Waltham, MA).

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).