Fig. 3.

Affinity chromatography and target identification. (A) Structures of positive (AM·reversine+) and negative (AM·reversine−) affinity matrices. (B) Silver staining and Western blot analysis of proteins retained by affinity supports. (C and D) Reversine inhibits the enzymatic activity of active MEK1 in a dose-dependent fashion as indicated by ERK2-phosphorylation (C) and reversine inhibits the ATPase activity of myosin II (heavy chain) in vitro (D). (E) Reversine (20 nM) blocks MEK-dependent ERK1/2-phosphorylation in C2C12 cells. C2C12 cells were treated with 20 nM reversine for 1 h, and cells were lysed and analyzed by Western blot. DMSO was used as a negative control. U0126 (10 μM) was used as a positive control. (F) Overexpression of MEK1 or NMMII can partially block reversine's activity. C2C12 myoblasts (plated at 6,000 cells per cm² in GM) transiently transfected with MEK1 or NMMII (with FuGENE6) were cultured in the presence of 20 nM reversine for 48 h. The compound was then removed, and cells were cultured in OI or AI conditions for an additional 6 days and analyzed by histocytochemistry. Empty vector was used as a negative control (Mock). (G) C2C12 myoblasts transfected with siNMMIIs (with X-treme) and cultured in the presence of 10 μM U0126 in GM for 48 h gain multipotency. C2C12 myoblasts (plated at 6,000 cells per cm² in GM) transiently transfected with siNMMIIs were cultured in the presence of 10 μM U0126 for 48 h. The compound was then removed, and the cells were cultured in OI or AI conditions for an additional 6 days and analyzed by histocytochemistry. Cells transfected with NTsi and treated with DMSO were used as a negative control. Red, ALP, Oil red O; blue, HT.