Phosphorylation of p62 in the brains of patients with AD. (a) Frozen tissues (the temporal cortex) of patients with AD and controls were lysed and subjected to SDS-PAGE. HeLa cells were treated with Baf, and used as a positive control. P-S349 is detected in almost all patients with AD but is only faintly detected in controls. The P-S403 signal is visible in all samples except for a control subject (case 6). An anti-oligomer antibody detects smear bands in some patients with AD. The approximately 100 kDa bands appear to be non-specific (*). (b) The ratio of P-S349 or P-S403 levels against β-actin was measured. (c) Quantitative data show that the level of P-S349, but not of P-S403, is significantly increased in AD compared with controls (*p < 0.01). The value in AD is defined as 100%. (d) The ratio of P-S349 or P-S403 levels against p62 was measured. (e) The level of P-S403, but not of P-S349, is significantly decreased in AD compared with controls (*p < 0.05). (f) Frozen tissues were obtained from the temporal cortex of patients with AD and control subjects (n = 3 in each group). Sequential biochemical fractionation shows that P-S349 is detected in fractions 1 and 5 (f1 and f5) in AD, and f3 and f5 in controls. Note that the signal is enhanced in f1 in AD samples compared with controls. Instead, the signal is weaker in f5 of controls than that of AD samples. P-S403 is distributed in f1, f3 and f5 in AD, and f1 and f5 in controls. Similar results are observed in other samples of AD and control cases. The CHAPS-soluble fraction (f4) has a lower quantity of proteins compared with other fractions. Acetylated histone is used as one of the nuclear proteins. Non-specific (*).