Anti γ adaptin

MNT-1 cells grown on coverslips were incubated for 3 days in medium containing peptides (10 µM). Media containing peptides were renewed every day. Coverslips fixed in 100% glacial methanol for 5 s were washed 5 times in distilled water and incubated in PBS/1 mg/mL BSA (incubation buffer, IB). Fixed cells were incubated for 1 h with mouse monoclonal anti–γ-adaptin (Sigma-Aldrich, clone 100/3) and rabbit polyclonal to KIF13A (Bethyl Laboratory, Inc., A301-077A) diluted in IB, washed three times in IB, and incubated with the corresponding secondary antibody (Alexa Fluor, Invitrogen) for 30 min. Cells were washed twice in IB and once in PBS before mounting the coverslips in DABCO medium (Invitrogen) and examining on an Eclipse 80i Upright Microscope (Nikon, Tokyo, Japan) equipped with a CoolSNAP HQ2 CCD Camera, a Piezo Flexure Objective Scanner and 100× Plan Apo objective (1.4 NA CFI).

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.