Journal: The Journal of Biological Chemistry
Article Title: Host Protein Ku70 Binds and Protects HIV-1 Integrase from Proteasomal Degradation and Is Required for HIV Replication *
Figure Lengend Snippet: IN is degraded through the Lys 48 -linked polyubiquitination proteasomal pathway, and Ku70 protects IN by reducing the overall ubiquitination level in the cells and partially blocking ubiquitination of IN and its bound cellular proteins. A , IN is degraded through Lys 48 -linked polyubiquitination. 293T cells were mock-transfected or cotransfected with GFP-IN and HA-Ubwt, HA-UbK48R, or HA-UbK63R for 48 h, as indicated. The ubiquitination levels of IN and IN-associated proteins were checked by IP with anti-GFP antibody and anti-HA antibody in a WB ( middle panel ). The same membrane was reprobed with anti-GFP antibody to detect GFP-IN expression in each sample ( top panel ). About 5% of the cells prior to co-IP were lysed in 0.5% Nonidet P-40, and the protein contents were subjected to WB to detect total HA-Ub levels in the cells using anti-HA antibody ( bottom panel ). B , Ku70 protects IN from degradation by targeting the cellular ubiquitin-proteasome pathway and reducing ubiquitin binding to IN. 293T cells were mock-transfected ( lane 1 ) or cotransfected with GFP-IN, HA-Ubwt and T7 vector, or T7-Ku70wt and T7-Ku70(1–430) ( lanes 2–4 ). The co-IP assay was done at 48 h post-transfection using anti-GFP antibody to pull down IN and its associated proteins and using immunoblotting with anti-HA antibody to determine the ubiquitination level of IN and its associated proteins ( second panel ). The same membrane was reprobed with anti-GFP antibody to examine GFP-IN expression ( first panel ). Simultaneously, equal amounts of total cellular proteins (about 5% of the total cell lysates) were resolved on a SDS-PAGE gel and immunoblotted with anti-HA and anti-T7 antibodies to determine the expression levels of the transfected protein expressors ( third and fourth panels ). β-Actin was used as a loading control ( bottom panel ). C , reduction of ubiquitin level by Ku70 is independent of the Lys 48 - and Lys 63 -linked polyubiquitination proteasomal pathway. 293T cells were transfected with HA-Ubwt/mut with T7-Ku70 or T7 vector for 48 h. Cells were lysed and analyzed for HA-Ub expression using anti-HA antibody in a WB. On the same membrane, β-actin was used as a protein-loading control. D , endogenous ubiquitin levels were increased in Ku70-down-regulated cells. C8166 T cells were transduced with empty vector or lentiviral vector expressing an shRNA against human Ku70 and selected with 1 μg/ml puromycin. After 1 week of selection, equal amounts of control or stable Ku70-KD cell lines were collected and lysed. The expression levels of ubiquitin, Ku70, and β-actin were assessed by WB using specific antibodies.
Article Snippet: To examine the IN/Ku70 interaction in HIV-1-infected cells, HIV-1 (HxBru or HxBru-IN-HA)-infected C8166 T cells were lysed with 0.25% Nonidet P-40 and immunoprecipitated with anti-HA antibody followed by WB with anti-Ku70 antibody to detect IN-bound Ku70.
Techniques: Blocking Assay, Transfection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Plasmid Preparation, SDS Page, Transduction, shRNA, Selection