induces caspase 3-dependent apoptosis in PC12-GFP and PC12-SH2B1β cells. PC12-GFP and PC12-SH2B1β cells were treated with 0, 100, 200, 300 μM H2O2 for 18 h. Equal amounts of proteins from the lysates were resolved with SDS-PAGE and immunoblotted with (A) anti-caspase 3 (upper panel), or (B) anti-PARP antibody. ERK levels were used as loading controls. For % cells with anti-active caspase 3 staining, cells were treated with 100 or 200 μM H2O2 for 18 h and then subjected to immunofluorescence staining using anti-active caspase 3 antibody (A, lower panel). Percentages of active caspase 3-positive cells were counted from 145-211 cells/condition. (C) Hippocampal neurons from E18 embryos were transiently transfected with GFP, GFP-SH2B1β or GFP-SH2B1β(R555E) and then treated with H2O2 for 18 h. Cells were then subjected to immunofluorescence staining with DAPI (shown in blue) to mark the nucleus. Green fluorescence (GFP) showed the transfected cells. Boxes mark the nucleus and arrows point to the neurites. Enlarged images of the nucleus and neurites are shown on the right panels.