Changes in Phospho-MAP/Tau during Neuronal Stress Figure 1. ATP reduction causes persistent phosphorylation of 12E8 epitope and dephosphorylation at other sites. (A) Epitope map of tau antibodies in human and chicken brain [38]. Schematic representations of the longest tau isoforms are shown (441 amino acid residues in human tau, 463 in chicken). The phosphorylation-dependent anti-tau antibodies (AT270, AT8, S214, AT100, AT180, 12E8, S396 S404 and S422) are shown with their corresponding target residues (S = Serine, T = Threonine). Positions of the various alternatively spliced inserts which give rise to 6 different isoforms in humans and 5 in chick are shown in blue. Green boxes denote tandem amino acid sequence repeats, which constitute the MTBD. (B) Representative Western blots of extracts from cell cultures treated in duplicate with 1 mM AM for 10, 30, 60 or 120 min. (C) Quantification of the Western blots shown in (B) reveal rapid dephosphorylation of AT8, S396 and AT270 epitopes (respectively declining to 1660.03%, 2369% and 4369% of control values; mean %6min/max intensities). By 30 min, phosphorylation at epitope S404 had also markedly declined (25611%) while epitopes S422 and AT180 exhibited a more moderate decline in phosphorylation (reduced to 5563% and 65620% at 30 min). Total tau was observed to decrease transiently (4165% at 30 min) before returning to control levels (98619%) at 120 min. All antibodies were probed on fresh blots and normalized to GAPDH loading control bands. (D) By contrast, phosphorylation of the 12E8 epitope increased markedly. To ascertain a phosphorylation:total tau ratio, blots probed for 12E8 or AT8 (for comparison) were stripped and reprobed for total tau (polyclonal rabbit antibody). Whereas the AT8:total tau ratio decreased rapidly, the 12E8:total tau ratio increased at 30 min and peaked at ,200% after 60 min. Error bars in (C and D) indicate the maximum and minimum intensities. (E) Quantification of persistent phosphorylation at the 12E8 epitope (normalized to GAPDH) was significant with a peak of .200% at 60 min and sustained phosphorylation (,150%) at 120 min (n = 4; *p,0.05). Error bars = SEM. doi:10.1371/journal.pone.0020878.g001 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20878