Anti ha polyclonal antibody

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).

For Western blotting, transfected cells were lysed in buffer containing 100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 20% β-mercaptoethanol and boiled for 5 min. Then, proteins in cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Subsequently, membranes were blocked for 1 h in PBS containing 5% dried skim milk and 0.1% Tween-20 and incubated with an anti-HA polyclonal antibody (Sigma, Stockholm, Sweden) or an anti-actin monoclonal antibody (Sigma) at 4 °C overnight. Horseradish peroxidase (HRP) secondary antibodies (Merck life science, Darmstadt, Germany) specific for either mouse or rabbit immunoglobulins (Ig) were used to detect bound primary antibodies. Proteins in the membranes were detected with a SuperSignal West Femto maximum-sensitivity chemiluminescent substrate kit (Thermo Scientific) following the manufacturer’s instructions.

For confocal laser scanning microscopy assays, DF1 cells grown in glass-bottomed culture dishes (NEST Biotechnology Co. LTD, Wuxi, China) were co-transfected with pCSGalNAcT2 and pGtVP2 as described above. Single transfections with 4 μg pCSGalNAcT2 or 4 μg pGtVP2 were performed as controls. At 36 h post-transfection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% bovine serum albumin (BSA). Then, cells were incubated with the anti-FLAG monoclonal antibody and the anti-HA polyclonal antibody (Sigma) followed by addition of the corresponding FITC- or TRITC-conjugated secondary antibody for 1 h at room temperature. After washing three times with PBST containing 0.05% Tween-20, cells were stained with DAPI (Beyotime Institute of Biotechnology) for 10 min and analyzed using a Leica SP2 Confocal system (Leica Laser Technik, Heidelberg, Germany). Additionally, to determine whether the exogenous CSGalNAcT2 was expressed and located in the Golgi apparatus, we used confocal microscopy to evaluate the location of the exogenous CSGalNAcT2 and the endogenous GOLGA2 protein, a known marker for the Golgi apparatus, using GOLGA2 antibody (Sigma).