Amersham protran western blotting nitrocellulose membranes

Transfected HEK293T cells and 729 HTLV-1.wt or HTLV-1.mEnhancer producer cell clones were lysed in RIPA buffer with protease inhibitor cocktail (cOmplete™, Mini Protease Inhibitor Cocktail, Roche Diagnostics GmbH, Mannheim, Germany). Total protein was quantitated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL) and FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA). Protein was loaded in equal amounts on 4–20% Mini-PROTEAN^® TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA) and transferred onto Amersham™ Protran^® Western blotting nitrocellulose membranes (Cytiva, Marlborough, MA). Membranes were blocked with 5% milk in PBS with 0.1% Tween-20 and incubated in the following primary antibodies: anti-Tax (rabbit anti-sera), anti-Hbz (rabbit anti-sera), and anti-β-actin (1:5,000; A2228, Sigma-Aldrich, St. Louis, MO). Membranes were incubated in appropriate anti-rabbit or -mouse IgG (H+L) HRP conjugate secondary antibodies (1:5000; W401B or W402B, Promega, Madison, WI). Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL) was used to develop the membranes and images were captured using an Amersham Imager 600 (GE Healthcare Life Sciences).

Transfected HEK293T cells and 729.B parental, WT, or mutant producer cell clones were lysed in NP-40 buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], and 1% NP-40) with protease inhibitor cocktail (cOmplete, Mini Protease Inhibitor Cocktail, Roche Diagnostics GmbH, Mannheim, Germany). Total protein was quantitated using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL) and FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA). Protein (10 μg) was loaded in equal amounts on 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio- Rad, Hercules, CA) and transferred onto Amersham Protran Western blotting nitrocellulose membranes (Cytiva, Marlborough, MA). Membranes were blocked with 5% milk in PBS with 0.1% Tween-20 and incubated with anti-Hbz (rabbit anti-sera [19 (link)]; 1: 500) and anti-β-actin (1:5,000; A2228, Sigma-Aldrich, St. Louis, MO). Membranes were incubated in appropriate anti-rabbit or -mouse IgG (H+L) HRP conjugate secondary antibodies (1:5,000; W401B or W402B, Promega, Madison, WI). Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL) was used to develop the membranes and images were captured using an Amersham Imager 600 (GE Healthcare Life Sciences).