Mini protean tgx precast 4 to 20 gels

Cells were lysed in NP-40 lysis buffer containing a protease inhibitor cocktail (Millipore Sigma) and quantitated using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equivalent amounts of protein were separated in Mini-Protean TGX precast 4 to 20% gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes. Membranes were blocked in phosphate-buffered saline (PBS) containing 5% milk plus 0.1% Tween^® 20 and incubated with the primary antibody. The following primary antibodies were used: HBZ (1:1000) [14 (link)], APH-2 (1:1000) [74 (link)], S-tag (1:1000; Abcam), Tax (1:1000), YBX1 (1:5000, Bethyl A303-231A), myc (1:1000; Abcam ab32), and β-actin (1:5000; Millipore Sigma). The secondary antibodies used were horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse immunoglobulin antibodies (1:5000; Santa Cruz Biotechnology). Membranes were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and imaged using an Amersham Imager 600 (GE Healthcare Life Sciences, Piscataway, NJ, USA). Densitometric data were calculated using the ImageQuant TL program (GE Healthcare Life Sciences).

Cell lysates were harvested in NP-40 lysis buffer containing protease inhibitor cocktail (Roche Applied Bioscience) and quantitated using a Pierce bicinchoninic acid protein assay kit (Fisher Scientific). Equivalent amounts of protein were separated in Mini-Protean TGX precast 4 to 20% gels (Bio-Rad Laboratories, Hercules, CA, United States) and transferred to nitrocellulose membranes. Membranes were blocked in phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20 and incubated with primary antibody. The following antibodies were used: anti-S-tag (1:1000; Abcam, Cambridge, MA), anti-UBR5 (1:1000; Cell Signaling Technology, Danvers, MA, United States), anti-HBZ (1:1,000), anti-APH-2 (1:1,000), anti-FLAG clone M2 (1:1,000; Agilent Technologies, Santa Clara, CA, United States), anti-ubiquitin (1:250; Santa Cruz Biotechnology), anti-β-actin (1:5,000; Sigma–Aldrich), and anti-α-Tubulin (1:250; Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse immunoglobulin antibodies (1:5,000; Santa Cruz Biotechnology). The blots were developed using an ECL Western Blotting Substrate (Fisher Scientific). Images were taken using an Amersham Imager 600 imaging system (GE Healthcare Life Sciences), and densitometric data were calculated using the ImageQuant TL program (GE Healthcare Life Sciences).