Bafilomycin a1 baf

To evaluate the binding and uptake of virus particles, DCs were incubated for 2 h at 37°C and at 4°C. Cells were collected, counted, and resuspended in distilled water and stored at −20°C. To assess the number of DNA copies, viral DNA was extracted with a Qiagen EZ1 Virus Mini 2.0 extraction kit. A specific PCR was performed using a Quantifast ROX vial kit (Qiagen, Sweden) or Takyon No ROX Probe Master Mix dTTP (Eurogentec S.A., Belgium) with HSV-2 gG forward primer (AGA TAT CCT CTT TAT CAT CAG CAC CA) and HSV-2 gG reverse primer (TTG TGC CAA GGC GA) and a probe (CAG ACA AAC GAA CGC CG) (32 (link)).
To determine whether the HSV-2 infection of DCs was dependent on acidification, we used the acidification inhibitors NH₄Cl (40 mM; Sigma) and bafilomycin A1 (BAF) (50 nM; Sigma). Additionally, the requirement for endocytosis was assessed using cytochalasin D (CCD) (10 μM; Sigma); clathrin-mediated endocytosis was assessed using chlorpromazine (CP) (6.25 μg/ml; blocks clathrin-mediated entry; Sigma), and protein transport was assessed with monensin (Mon) (4 μl/ml; BD). All the agents were used 30 min before infection of the DCs.

To silence the function of ATG5, XTC.UC1 cells were transfected with Atg5 siRNA (100 nM) using Lipofectamine^® RNAiMAX (Invitrogenm Carlsbad, CA, USA) according to the manufacturer's protocol. The medium was replaced after 6 hr and cells were incubated for a further 48 hr. Knockdown of ATG5 was confirmed for each experiment by performing western blot analysis with anti-ATG5 antibody (Cell signaling). XTC.UC1 cells were cultured on coverslips in 12-well plates for 48 hr after seeding. Thereafter, cells were treated with 5 mM and 10 mM 3-methyladenine (3-MA; Sigma-Aldrich) or 10 and 20 nM bafilomycin A1 (BAF; Sigma-Aldrich) for 0, 12, 24, and 36 hr. After immunofluorescent staining, the stained slides were observed under a confocal microscope (Olympus Corp.).

Biotin (Sigma-Aldrich, cat. B4501) and 4-PBA (Sigma-Aldrich, cat. P21005) were dissolved in distilled water and used at 100 μM and 1 mM final concentrations, respectively. MG132 (Sigma-Aldrich, cat. M7449), Bafilomycin A1 (BAF) (Sigma-Aldrich, cat. B1793), and Ko143 (Sigma-Aldrich, cat. K2144) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, cat. 276855) and applied at 2 μM, 10 nM, and 1 μM final concentrations, respectively. For solvent controls, distilled water and DMSO were used accordingly.