Horseradish peroxidase conjugated protein a

Immunoblots were developed using an ECL camera (Fusion Pulse 6, Vilber). Autoradiographs were analyzed and quantified with a Typhoon 9410 (GE Healthcare Life Sciences). Quantitative analysis of immunoblots was performed using ImageJ 1.52a (National Institutes of Health, USA). Statistical analysis was performed with GraphPad Prism 6.07. Models of ribosomal particles were generated with PyMOL 2.2.0. Polyclonal antibodies directed against Srp54^{42 (link)} (1:4000), Sgt2^{24 (link)} (1:5000), Get4^{24 (link)} (1:5000), Get5^{24 (link)} (1:5000), Get3^{24 (link)} (1:5000), Rpl4^{24 (link)} (1:10,000), Rpl24^{42 (link)} (1:10,000), Rpl35^{60 (link)} (1:10,000), Rpl31^{60 (link)} (1:5000), Rpl26^{60 (link)} (1:2000), Rps9^{42 (link)} (1:5000), Pgk1^{75 (link)} (1:2000), Kar2^{76 (link)} (1:2000), Sse1^{42 (link)} (1:20,000) (Eurogentec, Rospert lab antibody collection), and PAP (Sigma ID P1291, 1:5000) were raised in rabbit. α-FLAG (Sigma F3165, 1:2000) and α-His (Qiagen 34660, 1:2000) are monoclonal antibodies. Horseradish peroxidase-conjugated protein A (ThermoFischer Scientific 101023, 1:5000) was employed as a secondary reagent to detect primary antibodies. Dilutions employed for immunoblotting experiments are given in brackets. References, which include validation data of the different antibodies are indicated.

B78-nectin-1 or HaCaT cells (for internalization) or Vero cells (for penetration) were pretreated with MLN4924 or MG132 diluted in DMEM for 20 min at 37°C. Medium was replaced with ice-cold, carbonate-free, serum-free DMEM supplemented with 20 mM HEPES and 0.2% bovine serum albumin (binding medium). Cells were chilled for 10 min at 4°C on ice. HSV-1 KOS was added (~100 PFU/well) in ice-cold binding medium containing drug for 1 h at 4°C on ice. Cells were rinsed with ice-cold PBS, and warm complete DMEM was added. Cultures were returned to 37°C. At the indicated times p.i., cells were treated with warm sodium citrate buffer (pH 3.0) for 3 min at 37°C to irreversibly inactivate extracellular virions (112 (link)). Complete DMEM was added, and the infection was allowed to continue at 37°C. At 18 to 24 h p.i., cells were fixed with methanol-acetone (2:1). Cells were stained with rabbit polyclonal antibody to HSV (HR50; Fitzgerald Industries, Acton, MA) followed by a horseradish peroxidase-conjugated protein A (Thermo Fisher). Finally, 4-choloro-1-naphthol substrate (Sigma) and H₂O₂ catalyst (VWR International, Inc., Radnor, PA) were added to visualize plaques.

HSV-infected Vero cells were incubated at 37°C and 5% CO₂ for 18 to 24 h. Cells were fixed with methanol-acetone (2:1), dried, and stained with rabbit polyclonal antibody HR50 to HSV (Fitzgerald Industries, Acton, MA) and with horseradish peroxidase-conjugated protein A (Thermo Fisher Scientific). 4-Choloro-1-naphthol substrate (Sigma) and H₂O₂ catalyst (VWR International, Inc., Radnor, PA) were added to visualize plaques.