Protein g sepharose 4b

In order to detect protein-protein interactions, transfected 293 T cells were lysed with PBS containing 1% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich). In order to detect protein ubiquitination, cells were lysed with RIPA buffer (10 mM Tris pH7.8, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 5 mM EDTA) containing protease inhibitor cocktail. The immunoprecipitation assay was performed as described elsewhere. In brief, cell lysates were homogenized with 20 strokes of pipetting and then chilled on ice for 20 min. After centrifugation at 15,000 × g for 5 min, the supernatants were clarified by ultra-centrifugation (100,000 × g for 1 h at 4 °C, OptimaMAX-XP, Beckman Coulter, Brea, CA.). The supernatants were precleared with Protein-G Sepharose 4B (GE Healthcare, Little Chalfont, England) for 1 h at 4 °C. After brief centrifugation, the supernatants were immunoprecipitated with specific antibodies binding Protein-G Sepharose 4B overnight at 4 °C. Immunocomplexes were washed 6 times with buffer and analyzed by Western blotting.

COS cells, BAF cells or mouse T lymphocytes were lysed in buffer (1% Nonidet P-40, 150 mM NaCl, 25 mM Tris-HCl (pH 7.4), 10% glycerol, 2 mM MgCl₂, 1 mM phenylmethylsulfonylfluoride, 1 mM leupeptin and 0.1 mM aprotinin). For membrane fractions, the cells were washed in RPMI and resuspended on ice in a hypotonic buffer. Cells were sheared, and nuclei and unbroken cells were removed using low-speed centrifugation. The remaining supernatant was recentrifuged, and the cytosolic fraction (supernatant) was removed. The remaining pellet (membrane fraction) was washed twice with hypotonic buffer and finally resuspended on ice in lysis buffer44 (link). The lysates were precleared at 4 °C for 1 h with protein G-Sepharose 4B (GE Healthcare). Precleared lysates were immunoprecipitated with antibodies and protein G-Sepharose4B. The beads were washed four times with lysis buffer. Cell lysates or immunoprecipitates were subjected to immunoblotting3 (link).

Cells were harvested, washed with PBS, and lysed in a lysis buffer containing 50 mM tris-HCl pH 7.5, 150 mM NaCl, 100 mM NaF, 10 mM EGTA, 1 mM Na₃VO₄, 5 μM ZnCl₂, 10% glycerol, 1% triton X-100, and a proteinase inhibitor cocktail (Nacalai Tesque) for 30 min at 4°C. The lysates were then centrifuged, and the obtained supernatants were pre-cleared by incubating them with Protein G Sepharose 4B (GE Healthcare Life Sciences) for 1 h at 4°C. After brief centrifugation, the supernatants were reacted with anti-GFP antibodies at 4°C overnight, and then Protein G Sepharose beads were added and incubated with rotation for 1 h at 4°C. Immunoprecipitates were collected by brief centrifugation, and the mixtures were washed five times with washing buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% triton X-100, and analyzed by immunoblotting.

For PAGE, proteins were separated on NuPAGE gels containing a 4–12% polyacrylamide gradient (4–12% Bis-Tris gel; Novex-Thermo Scientific, Waltham, MA, USA) by using an MES buffer system (Novex-Thermo Scientific), according to the manufacturer’s instructions. The gels were stained with Coomassie Brilliant Blue R-250 and destained with 5% acetic acid. For immunoblotting, the proteins were electrophoretically transferred to a polyvinylidene fluoride membrane (FluoroTrans W; Pall, New York, USA) in a wet/tank blotting assembly. After transfer, the membrane was treated with a blocking solution containing 2.0% BSA (Roche) and 2.0% casein-derived blocking reagent (BlockAce; WakenBtech, Kyoto, Japan) in Tris-buffered saline for 2 h. The primary antibodies, mAbCML-1 and mAbCML-10, which were previously purified on protein G Sepharose 4 B (GE Healthcare, Chicago, IL, USA), were used at a dilution of 1:250–1:1000. For the secondary antibody, alkaline phosphatase-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) was used. Phosphatase activity on the membrane was visualized using a SIGMAFAST Fast Red TR/Naphthol AS-MX Tablet (Sigma-Aldrich) staining kit.

Each of the cell lines were seeded on six-well plates (one plate for each cell line) and were infected with lentiviruses expressing HCV core. After 48 h, the cells were collected, and the cell lysates were incubated with anti-HA antibody (HA.11, Covance, 1 μl per sample) at 4 °C for 90 min and then further incubated with Protein G Sepharose 4B (GE Healthcare) at 4 °C for 90 min. The beads were washed three times with lysis buffer, boiled at 95 °C for 5 min, and subjected to western blotting.