Journal: Journal of Virology
Article Title: Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1
Figure Lengend Snippet: Identification of mouse Sur2 as a specific MAV-1 E1A-interacting protein. (A) Large-scale GST-mE1A pulldown assay. Lane 1, protein molecular size standards; lanes 2 to 4, purified GST, GST-mE1A, and GST-Cter1 fusion proteins, respectively, were bound to glutathione-Sepharose beads without mixing with any mammalian cell nuclear extracts (−); lanes 5 to 7, nuclear extracts from 5 × 10 8 mouse MBMECs were precleared sequentially against glutathione-Sepharose beads and GST beads, and then an equal amount of the precleared mammalian nuclear extracts was added (+) to GST beads, GST-mE1A beads, and GST-Cter1 beads, respectively. Beads were washed, and the bound proteins were eluted off the beads by boiling in protein loading buffer. Proteins binding specifically to GST-mE1A were identified by comparing lane 6 with lanes 3 and 5. The arrowhead shows Sur2 protein. An independent duplicate analysis of both MBMEC and 3T6 cell lines gave similar results, with identification of Sur2 (data not shown). (B) Sur2 protein interacts with GST-mE1A (full-length E1A) in GST pulldowns. GST pulldown assays were performed by incubating the nuclear extracts from 3T6 cells (lanes 1 to 3) or MBMECs (lanes 4 to 6) with GST beads, GST-Cter1 beads, or GST-mE1A beads, as indicated. Monoclonal antibody against Sur2 (BD Pharmingen) (1:1,000) was used for Western blots. Whole-cell lysates (WCL) were used as a positive control (lane 8). Lane 7, protein molecular size standards. The arrowhead shows the Sur2 position. (C) MAV-1 E1A protein interacts with Sur2 in virus-infected cells. MBMECs were mock or MAV-1 infected at an MOI of 5 and harvested at 40 h postinfection. Normal rabbit serum or AKO7-147 (E1A) (both purified by DEAE Affi-Gel blue chromatography) was mixed with whole-cell lysates of MBMECs to carry out the immunoprecipitation. The immunoprecipitates were electrophoresed on 8% polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes. Monoclonal antibody against Sur2 was used in Western blots. The arrowhead shows the Sur2 position.
Article Snippet: The lysates were diluted in E1A binding buffer (125 mM NaCl, 50 mM Tris [pH 7.4], 0.1% NP-40) and then preabsorbed simultaneously against rabbit normal immunoglobulin G (IgG) and protein A-agarose beads (Pharmacia Biotech) by rocking at 4°C for 2 h. The lysates were then incubated with polyclonal antibody against MAV-1 E1A (AKO7-147) ( ) or rabbit normal IgG, followed by incubation with 30 μl of protein A-agarose (Oncogene Research Products).
Techniques: Purification, Binding Assay, Western Blot, Positive Control, Infection, Chromatography, Immunoprecipitation