Figure 3

(A) GUT-70 effects on Hsp90 client proteins. MCL cells were treated with GUT-70 (JVM-2, 5 μ M; Granta 519, 5 μ M; Jeko-1, 1 μ M; MINO, 5 μ M) for indicated times. Cells were subjected to lysis and analysed by western blot. Western blot images are representative results from three independent experiments. (B) GUT-70 competitively inhibited geldanamycin binding to the Hsp90α subunit. The competition of GUT-70 with FITC-labelled geldanamycin for binding to purified recombinant Hsp90α was measured. Fluorescence was measured at λex 485 nm and at λem 525 nm. Results shown are means±s.d. from three independent experiments. (C) GUT-70 induced protein ubiquitination. JVM-2 and MINO cells were treated with 5 μ M GUT-70 for 24 h, subjected to lysis, and immunoblotted for ubiquitin. Representative results are shown from three independent experiments. (D) Proteasome inhibitor bortezomib prevented GUT-70-mediated decreased expression of c-Raf. JVM-2 and MINO cells were treated with 5 μ M GUT-70 and/or 10 nM bortezomib for 24 h, subjected to lysis, and immunoblotted for c-Raf. Western blot images are representative results from three independent experiments.