FIG. 1.

Reduced VE-cadherin expression in the presence of K5. (A) Flow cytometry analysis of VE-cadherin expression in E-DMVECs 24 h after transduction with AdTet alone or AdTet together with AdK5 or AdK3 (MOI of 250). Cells were stained with VE-cadherin-specific antibody (BV9) or MHC-I-specific antibody (W6/32) followed by PE-conjugated secondary antibody. Gray shading, untransfected controls; dashed line, background staining with secondary antibody only; solid line, cells transduced with AdTet, AdK5, or AdK3. (B) Immunofluorescence analysis of E-DMVECs transduced with AdK5 and AdTet (MOI of 125). FLAG-tagged K5 was visualized with fluorescein isothiocyanate-conjugated anti-FLAG. VE-cadherin was stained with monoclonal antibody BV9 followed by PE-conjugated secondary antibody. (C) Biotinylation of cell surface proteins of uninfected E-DMVECs, E-DMVECs transduced with AdK5, or E-DMVECs with long-term latent KSHV infection. Biotinylated products, precipitated with streptavidin, were separated by SDS-PAGE and blotted with VE-cadherin- or TfR-specific antibodies. Note the increased expression of VE-cadherin in latently infected cells compared to that in uninfected cells, in contrast to the absence of VE-cadherin expression in K5-expressing cells. IB, immunoblot. (D) Immunoblot of VE-cadherin or calreticulin in total lysates from E-DMVECs transduced with AdTet alone or together with AdK5 as described in the legend to panel A. (E) Immunoblot analysis of anti-VE-cadherin immunoprecipitates (IP) with ubiquitin (Ub)-specific antibody. The expression of K5, cell lysis, and immunoprecipitation were the same as those for panel A except that ConA (50 nM for 3 h) was added where indicated. (F) Intracellular Myc staining of E-DMVECs transduced with AdTet and AdK5 with AdVE-cadherin or AdECTM (ECTM). Dashed line, background staining with secondary antibody only; solid line, cells transduced with AdK5 and AdECTM or AdVE-cadherin; gray shading, cells transduced with AdECTM or AdVE-cadherin only.