Anti flag polyclonal ab

Cells were lysed for 30 min on ice in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% SDS supplemented with a protease-inhibitor cocktail (Roche). Lysates were mixed with SDS-PAGE sample buffer and boiled for 5 min. Protein concentrations were determined with a BCA protein assay kit (Pierce; Thermo Fisher Scienti c, Waltham, MA, USA) using bovine serum albumin as a standard. Equal amounts of total protein were examined by western blotting using the following antibodies: anti-Flag monoclonal antibody (mAb; M2; Sigma-Aldrich), anti-Flag polyclonal Ab (Sigma-Aldrich), anti-β-actin mAb (Sigma-Aldrich), anti-HA mAb (MBL International, Woburn, MA, USA), anti-HIP1 mAb (Novus Biologicals, Littleton, CO, USA), anti-VprBP polyclonal Ab (Proteintech, Rosemont, IL, USA), horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Biosciences, Little Chalfont, UK), and HRP-conjugated goat anti-rabbit IgG (Amersham Biosciences). Signals were visualized after treatment with SuperSignal West Pico chemiluminescent substrate (Pierce; Thermo Fisher Scienti c).