Fig. 5.

Effects of RAB35 and MICAL1 on ROS-modulated Akt activity. a MCF-7 cells transfected with siRAB35 or siMICAL1, and protein levels of P-Akt and Akt were examined. Western blot bands corresponding to P-Akt were quantified and normalized against Akt levels. *: P < 0.05 in the cultures transfected with siRAB35 or siMICAL1 relative to the cultures with control siRNA. b MCF-7 cells were transfected with MICAL1 or RAB35 (CA) plasmids, and the total cellular proteins were extracted and analyzed for expressions of P-Akt and Akt by immunoblotting assays. *:P < 0.05 in the cells transfected with HA–MICAL1 or GFP–RAB35 (CA) relative to cells transfected with the corresponding vector. c MCF-7 cells were treated with 2 mM NAC for 1 h, and then the total protein extracts from cells were analyzed by immunoblotting assays for P-Akt and Akt expression. d Effect of NAC on cell invasion. After cultured in serum-free medium overnight, MCF-7 cells were pretreated with 2 mM NAC for 1 h, and then were stimulated with EGF (10 ng/mL) or 10 % FBS for 24 h. Quantifications of cells on the lower surface of the membrane were performed and shown. *: P < 0.05, **: P < 0.01 in the cultures with NAC relative to the cultures without NAC. e Invasion assays results showed that LY294002 and NAC delayed cell invasion in MICAL1-expressing MCF-7 cells. *:P < 0.05. **:P < 0.01