Figure 4.
Immunoblot analysis of the Al binding to PHFτ versus dephosphorylated PHFτ and autopsy-derived normal adult human τ as well as the effect of the chelating autoclave method on the Al binding to PHFτ. A-C: Aliquots of each of PHFτ (the Sarkosyl-PHFτ) (lanes 1–4 in A and lanes 1 and 2 in B), dephosphorylated PHFτ (lanes 3 and 4 in B and lanes 1 and 3 in C), and autopsy-derived normal adult human τ (lanes 5 and 6 in B and lanes 2 and 4 in C) were subjected to 10% SDS-PAGE and transferred to PVDF membranes. Each of the membrane strips was preincubated with control buffer without Al (lanes 1 and 3 in A, lanes 1, 3, and 5 in B, and lanes 1 and 2 in C) or buffer containing 10 mmol/L AlCl3 (lanes 2 and 4 in A, lanes 2, 4, and 6 in B, and lanes 3 and 4 in C) with shaking at room temperature for 1 hour, followed by probing with AT8 (lanes 1 and 2 in A), PHF1 (lanes 3 and 4 in A), T14 (lanes 1–6 in B), and Tau1 (lanes 1–4 in C). D: The immunoblot membrane strips containing PHFτ was preincubated with control buffer without Al (lane 1) or buffer containing 10 mmol/L AlCl3 (lanes 2–6) with shaking at room temperature for 1 hour. Following washing with TTBS after the incubation with Al, the membrane strips were incubated with control buffer without DFO (lanes 1 and 2), or treated by the autoclave procedure using deionized water (lane 3) or using DFO concentrations of 10 (lane 4), 30 (lane 5), and 100 mmol/L (lane 6). Each pair of lanes, 1–2 and 3–4 in A, 1–2, 3–4, and 5–6 in B, and 1 and 3 and 2 and 4 in C, as well as all lanes in D, are loaded with the same amount of the corresponding protein samples. Note that the incubation with Al totally abolished the immunoreactive bands and smear corresponding to those of PHFτ when probed with AT8 and PHF1 but not with T14. In contrast, no immunoreactive alterations were observed for dephosphorylated PHFτ and autopsy-derived normal adult human τ as revealed by T14 and Tau1. The Al-induced abolition of the immunoreactive bands of PHFτ was reversed by the chelating autoclave method in a DFO-concentration dependent manner. The position of Mr markers is indicated in kilodaltons at the left of each panel.