Anti β actin mab

Immunoblotting was performed according to standard procedures. Briefly, cells were washed in PBS and lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% [vol/vol] IGEPAL [Sigma], 1% [vol/vol] Na-deoxycholate, 0.1% [vol/vol] SDS, 1 mM DTT, 0.2 mM PMSF, 1 μg/ml leupeptin and pepstatin, 50 mM NaF, 0.1 mM Na-vanadate, and Complete protease inhibitor [Roche]). Equal amounts of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunoblot analysis was performed using mouse monoclonal antibody (MAb) anti-β-actin (Sigma), mouse MAb recognizing HCMV pUL83/pp65 (3A12; Abcam), and rabbit polyclonal anti-IκBα (sc-371; Santa Cruz). The proteins were visualized using peroxidase-coupled secondary antibodies (Dianova) and the ECL chemiluminescence system (Cell Signaling).

For immunoblotting analyses, 20E2 cells were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche Applied Science). Immunoblotting was performed as described previously^{74 (link)}. Primary antibodies used are: anti-myc tag mAb (Cell Signaling, Danvers, MA), C20 antibody, anti-β actin mAb (Sigma-Aldrich), anti-Phospho-Tyrosine mAb (Cell Signaling, Danvers, MA). Detection was performed with the Li-Cor Odyssey imaging system and quantitated with ImageJ 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% 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 Gn/Gc expression, 293FT cells (Thermo Fisher Scientific) were grown in 100 mm plates and calcium-transfected with the corresponding GPC encoding plasmid. 48 h later, cell surface proteins were labeled with biotin in order to separate the biotinylated (surface proteins) from non-biotinylated (intracellular proteins) fractions using a cell surface protein isolation kit (Pierce). For protein detection by western blot, primary anti-Gc monoclonal antibody (MAb) 2H4/F6 (Godoy et al., 2009 (link)), anti-Gn monoclonal antibody 6B9/F5 (Cifuentes-Muñoz et al., 2010 (link)) or anti-β-actin MAb (Sigma) were used at 1:2500 and subsequently detected with an anti-mouse immunoglobulin horseradish peroxidase conjugate (Thermo Fisher Scientific) 1:5000 and a chemiluminescent substrate (Pierce). All these antibodies were previously characterized concerning their reactivity with negative controls (Cifuentes-Muñoz et al., 2011 (link); Cifuentes-Muñoz et al., 2010 (link)). VLPs were harvested from supernatants of 293FT cells transfected with the pI.18/GPC wild type or the different mutant constructs at 48 h post-transfection and concentrated as previously established (Acuña et al., 2014 (link)). The amount of GPC encoding plasmid used for each mutant was adjusted in order to reach similar amounts of cell surface accumulation to wild type.