Horseradish peroxidase hrp conjugated goat anti mouse igg

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 an SDS-PAGE sample buffer and boiled for 5 min. Protein concentrations were determined with a BCA protein assay kit (Pierce; Thermo Fisher Scientific, 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 antibody (Sigma-Aldrich), anti-HA polyclonal antibody (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 Scientific).

Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membrane as described elsewhere [31 (link)]. The following antibodies were used: anti-Gag rabbit pAb (Bio Academia), anti-Vpr mouse mAb (Cosmo Bio Co., LTD), anti-FLAG M2 mouse mAb (Sigma-Aldrich), anti-GST rabbit pAb (Medical and Biological Laboratories Co., LTD), anti-GST goat pAb (GE Healthcare), anti-β-actin mouse mAb (Sigma-Aldrich), horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Biosciences), HRP-conjugated goat anti-rabbit IgG (Amersham Biosciences), and HRP-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology). The intensity of the protein bands was measured using an ImageJ v.1.50 software.

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% sodium dodecyl sulfate (SDS) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Lysates were mixed with SDS-PAGE sample buffer and boiled for 5 min. Protein concentrations were determined with a BCA protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Equal amounts of total protein were examined by Western blot. The following antibodies were used: anti-Flag MAb (M2) and anti-βnactin MAb (Sigma-Aldrich), anti-cleaved caspase-3 (Asp175) polyclonal antibody, anti-caspase-3 polyclonal antibody (Cell Signaling Technology, Beverly, MA), anti-GFP MAb (MBL, Nagoya, Japan), horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Bioscience, Uppsala, Sweden), and HRP-conjugated goat anti-rabbit IgG (Amersham Bioscience). Signals were visualized after the treatment of the membrane with SuperSignal West Pico chemiluminescent substrate (Pierce).