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. 2013 Jul 16;288(34):24691–24704. doi: 10.1074/jbc.M113.469692

FIGURE 3.

FIGURE 3.

Overexpression of SH3BP5 inhibits V642I-APP-induced death of mouse neuronal cells. A–D, F11 cells were cotransfected with the empty pFLAG vector (vector) or the pFLAG vector encoding mouse SH3BP5 (mSH3BP5) together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 72 h after transfection, they were harvested for microscopic analysis (A), Trypan Blue cell death assays (B), and WST-8 cell viability assays (C). Cell lysates were subjected to SDS-PAGE and immunoblot analysis with FLAG antibody (M2) and APP antibody (22C11) (D). E, PHNs were cotransfected with the empty pFLAG vector (vector) or the pFLAG vector encoding mSH3BP5 together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 48 h after transfection, the cells were immunostained with both the APP and FLAG antibodies. Nuclei were stained with DAPI. For the calculation of cell-death percentages in the cells that had been transfected with the two empty vectors, the percentages of morphologically apoptotic cells in total cells were counted. For the calculation of cell-death percentages in the cells that had been cotransfected with the empty vector and the V642I-APP-encoding vector, with the mSH3BP5-encoding vector and the empty vector, or with the V642I-APP- and mSH3BP5-FLAG-encoding vectors, the percentages of apoptotic cells in the cells that overexpressed V642I-APP, SH3BP5-FLAG, and both V642I-APP and mouse SH3BP5-FLAG, were counted, respectively. This study was performed with n = 3; 50–100 cells were counted per well. ***, p < 0.001; n.s., not significant.