Figure 3. Phosphorylation of H2A on T17 by Akt occurs during H₂O₂-induced cell death.

(a) PC12 cells (left) and primary cultured hippocampal neurons (right) were treated with 1 mM or 0.5 mM H₂O₂ respectively for 30, 60, and 120 min and immunoblotting was performed with the indicated antibodies. (b) Fixed hippocampal neuron cells were stained with Hoechst 33342 (blue), anti-pAkt antibody (green), and anti-Tau1 antibody (red). (c) PC12 cells were maintained without serum for 4 h, followed by H₂O₂ treatment. Fixed cells were stained with DAPI (blue) and anti-pAkt antibody (green). After H₂O₂ stimulation for 30 min, active Akt translocated from the cytoplasm into the nucleus. (d) Total nuclear proteins were isolated from PC12 cells treated with or without H₂O₂ and fractionated into cytoplasmic and nuclear proteins. Nuclear fractions were analyzed by immunoblotting with anti-pH2A antibody. PARP was used as a marker for the nuclear fraction. (e) PC12 cells were stimulated with 1 mM H₂O₂ for 30 min and cell lysates were used for immunoprecipitation with anti-H2A antibody. The association between endogenous Akt and H2A was enhanced by H₂O₂ treatment. (f) RFP-Akt constructs (active Akt T308DS473D or kinase-dead K179A) were co-transfected with GFP-tagged H2A-WT and mutant (H2A-T17A) into HEK 293T cells. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (g) GST-Akt and GFP-H2A constructs (WT and mutant H2A-T17A) were co-transfected into PC12 cells. After 24 h, cells were stimulated with 1 mM H₂O₂ for 30 min and cell lysates were used for co-precipitation with glutathione beads. The association between exogenous Akt and H2A-WT was enhanced by H₂O₂ treatment.