Lipid-linked oligosaccharides and other glycolipids were labeled in a cell-free system as described [17 (link),18 (link)]. Briefly, washed cell lysates were supplemented with 5 mM MnCl2 and 1 mM DTT, incubated with GDP[3,4-3H]Man (0.5 μCi/mL, 18.3 Ci/mmol; PerkinElmer Life Sciences) and 2 mM UDP-GlcNAc for 5 min. Tunicamycin (0.8 μg/mL, Boehringer Mannheim, Indianapolis IN) was added to facilitate the identification of the dolichol-linked N-glycan precursor. The reaction was continued after the addition of 1 mM GDP-Man (20 min at 27 °C) and then terminated by addition of CHCl3/CH3OH (1:1, v/v) to yield CHCl3/CH3OH/H2O in a 10:10:3 (v/v/v) ratio. Insoluble material was removed by centrifugation and organic supernatants dried under nitrogen. Lipids were extracted by addition of equal volumes of n-butanol and water, with the organic upper phase removed to a new tube. The lower aqueous phase was re-extracted twice with water-saturated n-butanol and the organic phases pooled and dried. Samples were then analyzed on silica gel 60 TLC plates (Merck) with plates developed in CHCl3/CH3OH/H2O (10:10:3, v/v/v), dried, and sprayed with En3Hance (DuPont) and exposed to film at −80 °C.
Tunicamycin
Manufactured by Roche
Tunicamycin is a laboratory product manufactured by Roche. It is a naturally occurring glycosylation inhibitor that blocks the first step of N-linked glycosylation in eukaryotic cells. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using tunicamycin
1
Synthesis and Analysis of Glycolipids
2
Radiolabeling of Lipid-Linked Oligosaccharides
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Lipid-linked oligosaccharides and other glycolipids were labeled in a cell-free system as described [17] (link), [18] (link). Briefly, washed cell lysates were supplemented with 5 mM MnCl2 and 1 mM DTT, incubated with GDP[3,4-3H]Man (0.5 µCi/mL, 18.3 Ci/mmol; PerkinElmer Life Sciences) and 2 mM UDP-GlcNAc for 5 min. Tunicamycin (0.8 μg/mL, Boehringer Mannheim, Indianapolis IN) was added to facilitate the identification of the dolichol-linked N-glycan precursor. The reaction was continued after the addition of 1 mM GDP-Man (20 min at 27 °C) and then terminated by addition of CHCl3/CH3OH (1:1, v/v) to yield CHCl3/CH3OH/H2O in a 10:10:3 (v/v/v) ratio. Insoluble material was removed by centrifugation and organic supernatants dried under nitrogen. Lipids were extracted by addition of equal volumes of n-butanol and water, with the organic upper phase removed to a new tube. The lower aqueous phase was re-extracted twice with water-saturated n-butanol and the organic phases pooled and dried. Samples were then analyzed on silica gel 60 TLC plates (Merck) with plates developed in CHCl3/CH3OH/H2O (10:10:3, v/v/v), dried, and sprayed with En3Hance (DuPont) and exposed to film at −80 °C.
3
ASFV Infection and AP-1 Interaction
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Cells were MOCK-infected or infected with ASFV E70 (MOI = 3). The infected cells were either treated or not with tunicamycin (Boehringer Mannheim) at 5 μg/ml, which was added after the virus adsorption. At 16 hpi cells were collected and lysed (25 mM Tris-HCl [pH7.5], 150 mM NaCl, 2 mM EDTA, 10 mM MgCl, 0.5% glycerol, protease inhibitors [0.8 μg/ml aprotinin, 0.8 μg/ml pepstatin and 0.8 μg/ml leupeptin], 1% Triton x-100). The extracts were incubated with a specific antibody against AP-1 (Anti-γ-Adaptin, Sigma-Aldrich) at a final concentration of 1.2 μg/ml overnight at 4°C. Protein G-Sepharose beads (Sigma-Aldrich) were added, incubated for 3 h at 4°C, and centrifuged. The beads were washed three times with wash buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM EDTA). The immunoprecipitates were mixed with SDS loading buffer and analyzed by 10% SDS-PAGE, followed by Western blotting.
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