Figure 6

SH2B1β reduces H ₂ O ₂ -induced nuclear distributions of FoxO1 and 3a. PC12-GFP and PC12-SH2B1β cells were incubated in serum-free medium overnight before 0, 100, or 200 μM H₂O₂ treatment for 10 min, without (A-B) or with 20 μM LY294002 (+LY) (C-D, G-H) or 20 μM U0126 (+U) (E-F, I-J) pretreatment for 30 min. The localization of FoxOs was determined via immunofluorescence staining using anti-FoxO1 (A, C, E, G, I) or anti-FoxO3a (B, D, F, H, J) antibody followed by Alexa Fluor 555-conjugated secondary antibody. Images were taken using inverted Zeiss Axiover 135 fluorescence microscope. Percentage of cells with fluorescence intensity of FoxO1 or FoxO3a in the nucleus higher than in the cytoplasm (N > C) was quantified. A total of 90-110 cells were counted for each condition. For inhibitor assays, results from PC12-GFP cells are shown on the left panels and those from PC12-SH2B1β cells are shown on the right.