Odyssey infra red imaging scanner and software

Protein extracts were obtained by cell lysis in M-PER (Pierce Biotechnology, Rockford, IL) and Halt protease inhibitor cocktail (Pierce Biotechnology). Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked with Blocking Buffer (LI-COR, Lincoln, NE) and incubated with specific antibodies. Protein signals were visualized using the Odyssey infra-red imaging scanner and software (LI-COR).

Cell pellets from transfection procedures were lysed in Mammalian Protein Extract Reagent (M-PER; Pierce/Thermo Scientific, Rockford, IL, USA). Following pre-cleaning by centrifugation, protein concentrations of cell lysates were determined by using Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Lysates equivalent to 25 μg of protein were separated on NuPAGE Bis-Tris (4-12%) gels (Invitrogen, Carlsbad, CA, USA) and transferred onto PVDF membranes. Membranes were blocked in Blocking Buffer (LI-COR, Lincoln, NE, USA) and incubated with specific antibodies against ERG splice variants (ERG MAb 9FY; Biocare Medical Inc., Concord, CA, USA), C-MYC (Epitomics, Burlingame, CA, USA) and GAPDH (Santa Cruz biotechnology, Santa Cruz, CA, USA). Membranes were washed in Tris-Buffered Saline + Tween 20 (TBST) before incubation with appropriate secondary antibodies (goat anti- Mouse IRDye 8000CW or goat anti-Rabbit IRDye 680CW, LI-COR). Signals of proteins detected were visualized and quantitatively measured using the Odyssey infra-red imaging scanner and software (LI-COR).

VCaP, LNCaP, and HEK293 cells were trypsinized and washed twice with PBS. VCaP cells were counted on a Coulter cell counter and aliquoted in appropriate quantities. Cells were pelleted by centrifugation and pellets were lysed in Mammalian Protein Extraction Reagent (M-PER; Thermo Scientific). Following pre-clearing by centrifugation (13,000 × g for 10 min), protein concentrations of cell lysates were determined by using Protein Assay Reagent (Bio-Rad, Hercules, CA). LNCaP and HEK293 cell lysates were spiked with purified recombinant ERG protein at the indicated concentrations, and then both sets of lysates were separated on NuPAGE Bis-Tris (4-12%) gels (Life Technologies) and transferred onto PVDF membranes. Membranes were blocked in Blocking Buffer (LI-COR, Lincoln, NE) and incubated with specific antibodies against ERG (ERG MAb 9FY; Biocare Medical Inc., Concord, CA), and GAPDH (Santa Cruz biotechnology, Santa Cruz, CA). Membranes were washed in Tris-Buffered Saline + Tween 20 (TBST) before incubation with appropriate secondary antibodies (goat anti-Mouse IRDye 800CW or goat anti-Rabbit IRDye 680CW, LI-COR). Signals of proteins detected were visualized and quantitatively measured using the Odyssey infra-red imaging scanner and software (LI-COR).