Okumura et al. 10.1073/pnas.0510518103. |
Fig. 5. ISG15 does not affect the Vps28 and Tsg101 interaction. 293T cells were cotransfected with Vps28-His, ISG15, and Tsg101 expression plasmids, as indicated. Twenty-four hours after transfection, cells were lysed. (A) Lysates immune blotted with Vps28, Tsg101, or ISG15 antibodies. (B) The Vps28-His was purified from the lysates on a colum of Ni 2+ agarose beads. Immunoblot analysis revealed that His-Vps28 interact with Tsg101 both in the presence and absence of ectopic ISG15. The ISG15 containing proteins pull down by His-Vps28 have not yet been identified.
Supporting Text
Experimental Procedures
Virus stock:
Viral stocks were made by transfection of pLN-43 DNA to 293T cells and medium was collected at 48 h after transfection. For virus purification, 27 ml of the virus-containing media from transfected cells was pelleted although 2 ml of a 20% sucrose cushion at 100,000 ´ g for 1.5 h. Virions were resuspended in TSE (0.1 M NaCl/1 mM EDTA/0.01 M Tris·Cl; pH 7.4) and centrifuged to equilibrium in 5 ml of a 20-60% sucrose gradient in TSE at 200,000 ´ g for 2.5 h. Virus-containing fractions were detected by immunoblotting using HIV Ig serum (AIDS Research & Reference Reagent Program, National Institute for Allergy and Infectious Diseases). Peak fractions were pooled, diluted with TSE, and pelleted through 20% sucrose at 100,000 ´ g for 2 h.Transducing Virus.
The vector (301-ISG), the helper plasmid (pCMVR8.2) and VSV encoding plasmid (pMDG) were transfected in the ratio 3:2:1 to 293T cells. Supernatants were collected at 48 h after tranfection, filtered although a 0.45-mm-pore-size filter, and VSV-G pseudotypes concentrated by ultracentrifugation at 12,000 ´ g for 60 min. Virus titers were determined on 293T cells, and the virus was stored at –80°C before uses.Immunoprecipitation and Western Blots.
Cells were lysed in lysis buffer (20 mM Tris·HCl/150 mM NaCl/10 mM EDTA/0.5% Nonidet P-40/PMSF, and proteinase inhibitor mixture). For the immunoprecipitation, 1 mg of protein was incubated for 30 min on ice with 20 ml of protein G-agarose (Invitrogen) in a total volume of 1 ml. The mixture was centrifuged at 1,000 × g for 5 min to preclear the lysate of proteins binding nonspecifically to the matrix. The precleared supernatants were incubated with specific antibody (2 mg/ml) for 1 h on ice, and then 20 ml of protein G plus-agarose was added to the immune complexes for an additional 1 h on ice. The bound proteins were pelleted at 1,000 × g for 5 min and washed with lysis buffer three times. The final pellets were resuspended in 40 ml of sample buffer and boiled for 5 min before loading on a 10% SDS/PAGE gel. The coprecipitated proteins were identified by Western blotting. For Western blot analysis, cells were lysed in buffer (20 mM Tris·HCl, pH 7.5/150 mM NaCl/1mM Na2EDTA/1 mM EGTA/1% Triton/2.5 mM sodium pyrophosphate and protease inhibitor mixture; Sigma, P8340), and the lysates were clarified by centrifugation at 10,000 ´ g for 10 min. Proteins (50 mg) were separated on 10 or 12% SDS-polyacrylamide gels and electrotransferred to the membranes (Bio-Rad). The membranes were blocked in 5% nonfat milk for 30 min at room temperature and then sequentially incubated with primary antibody for 30 min in blocking buffer and the horseradish peroxidase-conjugated secondary antibody (1:10,000 dilutions; Amersham Pharmacia).Results
Ectopic ISG15 does not inhibit Tsg101 and Vps28 interaction.
To determine whether ISG15 disrupts also the Vps28 and Tsg101 interaction we have cotransfected cells with His- Vps28 and Tsg101 in the presence and absence of ISG15. Immunoblotting of the cell lysates with the respective antibodies showed that all transfected plasmids were well expressed (Fig. 5). Ni2+ pull down of His-Vps28 revealed the physical association between His-Vps28 and Tsg101 that was not affected by ectopic ISG15.