Figure 4. sT targets SCF^Fbw7 complex to stabilize LT.

(A) LT interacts with the Fbw7 E3 ligase complex. HA-tagged Fbw7 (6 µg) was cotransfected with MCV LT (6 µg) into U2OS cells and immunoprecipitated with anti-HA antibody. LT interaction was detected with CM2B4 antibody. Negative controls for immunoprecipitation include transfection with LT alone or an empty vector. (B) sT interacts with the Fbw7 E3 ligase complex. HA-Fbw7 (6 µg) was cotransfected with wild type (sT.wt) or mutant (sT.LSD_91-95A) or empty vector (6 µg), followed HA immunoprecipitation and immunoblotting for sT using CM8E6 antibody. MCV sT, but not mutant sT (sT.LSD_91-95A) interacted with overexpressed HA-Fbw7. White arrowhead indicates immunoglobulin light chains (LC) at 25 kDa. (C) LT interaction with the Fbw7 E3 ligase complex is accentuated with MG132 treatment. Binding of MCV LT to Fbw7 was examined with lysates treated with MG132 (10 µM, 24 h). Both MCV LT and Fbw7 proteins are stabilized in 293 cells treated with MG132, and protein interactions are more readily detected as compared to vehicle treatment control. (D) sT interaction with the Fbw7 E3 ligase complex is also revealed with MG132 treatment. Fbw7 was cotransfected into 293 cells with wild type (sT.wt) or mutant sT (sT.LSD_91-95A) or empty vector. Cells were treated with MG132 (10 µM, 24 h) 24 hours after transfection, then harvested. CM8E6 was used for sT immunoprecipitation followed by Fbw7 detection using HA antibody. Fbw7 in cell lysates as well as following immunoprecipitation with sT is more readily detectable by MG132 treatment. (E) MCV sT inhibits Fbw7 interaction with MCV LT. LT, sT, Flag-c-Myc and HA-Fbw7 proteins, were expressed in 293 cells treated with MG132 (10 µM) for 24 hours. Cell lysates were then immunoprecipitated with anti-HA antibody and immunoblotted for LT or Flag-c-Myc. No immunoprecipitation is seen in negative control lanes transfected with HA expressing vector without Fbw7 (lane 1–3) but coimmunoprecipitation of Flag-tagged c-Myc (lane 4) and LT (lane 5) by HA-Fbw7 is evident. LT-Fbw7 interaction is inhibited when both LT and sT are coexpressed with HA-Fbw7 (lane 6), but not when LT is coexpressed with the LSD mutant (lane 7). White arrowhead indicates the immunoglobulin heavy chains (HC) at 50 kDa. See also Figure S3.