Treatment of Aβ induces the phosphorylation of p66Shc at Ser36 residues in SH-SY5Y cells. (A) SH-SY5Y and PC12 cells were treated with 30 µM Aβ25-35 or Aβ35-25 for 24 h in serum-free media containing N2 supplements. Trypan blue exclusion was used to determine cell death. Data are means ± SEM for three separate experiments performed in duplicate. *, P < 0.05 versus untreated cells. (B) Western blot analysis showing phosphorylated and total p66Shc in SH-SY5Y cells treated with 30 µM Aβ for the indicated times after a 16-h serum starvation period by using a specific anti–phospho-p66Shc (Ser36) antibody (top) and an anti-Shc polyclonal antibody (bottom). The blots are representative of three separate experiments. (C) At 7 DIV, cultures were treated with Aβ for 24 h, whereupon representative photographs were taken to illustrate the changes in cell morphology. (D) At 7 DIV, cells treated with Aβ25-35 or Aβ35-25 for 24 h and Hoechst 33342 labeling were used to determine cell death. Data are the means ± SEM of three separate experiments performed in duplicate. (E) Western blot analysis to detect p66Shc phosphorylation in 7 DIV cultures treated with 25 µM Aβ for the indicated times was performed by using a specific anti–phospho-p66Shc (Ser36) antibody (top) and an anti-Shc polyclonal antibody (bottom). The blots are representative of three separate experiments. (F) SH-SY5Y cells treated with 30 µM Aβ for 30 min after a 16-h serum starvation period. The cells were harvested and the lysates were either treated with PP2A1 or left untreated, and then sequentially blotted with a specific anti–phospho-p66Shc (Ser36) antibody (top) and an anti-Shc polyclonal antibody (bottom). The blots are representative of three separate experiments.