Figure 4

CBD of Cdc37 plays a crucial role for Tax stability in cells. (a) Co-transfection of LTR-Tax and pcDNA3-Flag-tagged Cdc37 (F-Cdc37) mutants. Lane 1: control pcDNA3 1 μg; lane 2: LTR-Tax 0.5 μg+pcDNA3 0.5 μg; lane 3: LTR-Tax 0.5 μg+F-Cdc37(1–378) wild type 0.5 μg; lane 4: LTR-Tax 0.5 μg+F-Cdc37(1–200) 0.5 μg; lane 5: LTR-Tax 0.5 μg+F-Cdc37(1–180) 0.5 μg; lane 6: LTR-Tax 0.5 μg+F-Cdc37(181–378) 0.5 μg; lane 7: LTR-Tax 0.5 μg+F-Cdc37(201–378) 0.5 μg. After 40 h transfection, HEK293 cells were lysed with 100 μl of lysis buffer, and the NF-κB-dependent luciferase activity was normalized with β-galactosidase value (upper panel). A portion of 10 μg of each lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the expression of Tax (middle panel) or Flag-tagged Cdc37s (lower panel) was detected by specific monoclonal antibodies. (b) Dose-dependent degradation of Tax by F-Cdc37 mutants. Lane 1: control pcDNA3 1 μg; lanes 2–11: 0.5 μg of LTR-Tax; lanes 3–5: plus 0.125, 0.25 and 0.5 μg of F-Cdc37(1–200); lanes 6–8: plus 0.125, 0.25 and 0.5 μg of F-Cdc37(1–180); lanes 9–11: plus 0.125, 0.25 and 0.5 μg of F-Cdc37(181–378). pcDNA3 was added to normalize the DNA amount. Tax (upper panel), Flag-tagged Cdc37s (middle panel) and tubulin (lower panel) were detected by specific monoclonal antibodies. (c) Cdc37’s CBD (amino-acid residues 181–200(ref. 26)) is required for Tax interaction. A portion of 0.5 μg of control pcDNA3 (lane 1) or LTR-Tax (lanes 2–9) was transfected (lysate-1, middle panel) and 0.5 g of control pcDNA3 (lanes 1 and 2), F-HSP90 (lane 3), wild-type F-Cdc37(1–378, lane 4), F-Cdc37(1–278, lane 5), F-Cdc37(1–200, lane 6), F-Cdc37(1–180, lane 7), F-Cdc37(181–378, lane 8) and F-Cdc37(201–378, lane 9) were transfected separately (lysate-2, lower panel). The expression of each protein in cell lysates was detected by specific monoclonal antibodies (middle and bottom panels). A portion of 200 μg of each lane’s cell lysates was mixed and subjected to Co-IP with 2 μg of rabbit anti-Flag antibodies, and each Co-IP complex was washed four times with Co-IP buffer, and following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Tax was detected by anti-Tax antibody (upper panel). (d) GFP two-hybrid binding assay between Cdc37 and Tax. HEK293 cells seeded on the six-well plates were transfected with phmKGN-MC-Tax and phmKGC-MN-Cdc37 or its mutant −Cdc37(N200) and −Cdc37(N180) by FugeneHD. After 48 h incubation, the transfected HEK293 cells were treated with Hoechst 34442 (Sigma) at the final concentration of 1 μM. Light and fluorescent (GFP and Hoechst 34442) microscopic observation and photography were performed by BZ-9000 Biorevo all-in-one fluorescence microscope (Keyence).