Figure 1

17-DMAG inhibits HSP90–Tax–IKK ternary complex formation and induces Tax degradation in ATL cells. (a) Co-IP of Tax and HSP90. Four million Jurkat or MT4 cells were lysed with Co-IP buffer, and 50 μg total of cell lysates were subjected to IP with 2 μg of rabbit polyclonal anti-HSP90 antibody, followed by immunoblot (IB) with mouse monoclonal anti-Tax antibody (upper panel). The amount of expressed Tax in each cell line was verified by IB with anti-Tax against 10 μg of cell lysates (lower panel). (b) 17-DMAG’s effects on Tax expression level and physical interaction between HSP90 and IKKβ in MT4 cells. Four million MT4 cells were treated with the indicated concentrations of 17-DMAG for 16 h. Co-IP and IB against immunoprecipitates or lysates were carried out as in panel a. (c) Ten micrograms of each cell lysate from MT4 cells treated with or without 2.5 μM of 17-DMAG for the indicated periods were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Tax (upper panel), HSP90 (middle panel) or tubulin (lower panel) expression was detected using monoclonal anti-Tax, anti-HSP90 or anti-tubulin antibodies. (d) Expression levels of Tax in 17-DMAG-treated MT4 cells. mRNAs were prepared from the same aliquots of MT4 cells described in panel c. mRNAs from 17-DMAG-untreated fractions (black bars) and 17-DMAG-treated fractions (2.5 μM, white bars) with indicated time courses were analyzed with the universal probes (Roche) and primers through the LightCycler PCR method according to the manufacturer’s direction. Comparison of Tax mRNAs with or without 17-DMAG was also indicated with a red line graph as a division of Tax mRNA with 17-DMAG/without 17-DMAG at each time point. (e) Four million MT4 cells were treated with 2.5 μM 17-DMAG for 24 h (lanes 2–6) and then additional 3 μM MG132 for 12 h (lane 3), 10 μM zVAD-fmk (lane 4) and 3-MA (lane 5) and 1 mM AICAR (lane 6) for 24 h. Lysates were prepared for IB of Tax and tubulin.