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Figure 2. Proline 394 within the kinase domain of LIMK1 confers recognition by Hsp90 and sensitivity to 17-AAG. A) Lysates of 293T cells expressing Flag-LIMK1 (LIMK1) or F-LIMK1-P394E (P394E) were incubated in the absence or presence of 1 µM 17-AAG for 2 h and subjected immunoprecipitation with anti-LIMK1 monoclonal antibody followed by Western blotting and probing with anti-Hsp90 and anti-LIMK1 mAbs. B) 293T cells expressing wt-LIMK1 or LIMK1-P394E were incubated in the absence or the presence of 1 µM 17-AAG for the period indicated (in hours). Cell lysates were subjected to Western blotting and probing with anti-Flag and anti-tubulin Abs. C) Summary of the data presented in (B). The experiments presented in (A) and (B) were performed twice and three times, respectively.
