Endo h

Transfected COS cells were scraped and lysed in lysis buffer (100 mM NaCl, 40 mM KCl, 1 mM EDTA, 20 mM HEPES, 10% Glycerol, 1% Triton-X, with Complete Protease Inhibitor from Roche) for 1 h at 4 °C. Lysates were cleared by 10 min centrifugation at 20,000 g. For deglycosylation, solubilized proteins were incubated with 250 U EndoH and/or 10 U PngaseF (Promega, France) at 37 °C overnight. Before separating proteins on a 10% polyacrylamide gel and blotting onto PVDF membrane (Hybond-P, Amersham Bioscience), lysates were boiled 5 min at 99 °C with Laemmli buffer containing reducing ß-Mercaptoethanol. Blots were blocked with 5% milk/PBS for 1 h and incubated with anti-TWIK2 polyclonal antibody (1:200, Alomone, Jerusalem, Israel) at 4 °C overnight or with anti-β-Tubulin monoclonal antibody (1:1000, clone TUB 2.1, Sigma-Aldrich, France) 1 h at RT. Secondary horseradish peroxidase coupled antibodies were diluted 1:10.000 and incubated 1 h at RT before detection by enhanced chemiluminescence reaction (ECL, Amersham Biosciences).

FLK-BLV or PK15-BLV were cultured in the absence or presence of 20 µM CMP5 for 48 h. Thereafter, the cell lysates were treated in the absence or presence of PNGase F (Promega, Madison, WI 53711-5399, USA) for 3 h or Endo H (Promega) overnight according to the manufacturer’s protocol (catalog# V483A and catalog #V4871), respectively.

To assess the glycosylation pattern of the UUKV glycoproteins, virus stocks purified through a 25% sucrose cushion were denatured and exposed to one of the five following treatments: 1,000 units of endoglycosidase H (Endo H; Promega), 5 units of peptide-N-glycosidase F (PNGase F), 0.005 units of α-2(3, 6, 8, 9)-neuraminidase, 0.003 units of β-1,4-galactosidase, and 0.05 units of β-N-acetylglucosaminidase (all enzymes from Merck Millipore) according to the manufacturer's recommendations. Samples were then analyzed by SDS-PAGE on a 4 to 12% or 10% Bis-Tris NuPAGE Novex gel (Life Technologies) and immunoblotting.

Radiolabeled cell pellets were resuspended in 100 μl of lysis buffer [20 mM Tris-HCl (pH 7.5), 1% SDS, 1 mM DTT, 1 mM PMSF, and 1× Protease Inhibitor Cocktail (Quartett)] and mixed with 100 μl of ice-cold glass beads. Cell suspensions were vortexed for 2 min twice, keeping the samples on ice for 1 min in between. Subsequently, the samples were incubated at 60°C for 15 min and centrifuged (6000 g, 1 min, 4°C). Supernatant fractions were mixed with 500 μl of immunoprecipitation buffer [15 mM Tris-HCl (pH 7.5), 0.1% SDS, 1% Triton X-100, and 150 mM NaCl], 1 μl of anti-HA antibody (MMS-101R; Biolegend) and 20 μl of prewashed protein G–agarose beads (Thermo Fisher Scientific, Pierce) and rotated at room temperature for 3 h. The agarose beads were washed twice with immunoprecipitation buffer, once with ConA buffer [500 mM NaCl, 20 mM Tris-HCl (pH 7.5) and 1% Triton X-100], and once with buffer C [50 mM NaCl and 10 mM Tris-HCl (pH 7.5)]. The beads were incubated with 50 μl of SDS sample buffer [50 mM DTT, 50 mM Tris-HCl (pH 7.6), 5% SDS, 5% glycerol, 50 mM EDTA, 1 mM PMSF, 1× Protease Inhibitor Cocktail (Quartett) and Bromophenol Blue] at 60°C for 15 min, followed by Endo H (Promega) treatment at 37°C for 1 h. Protein samples were then loaded onto SDS–PAGE gels and separated by electrophoresis.