Figure 5.

Phosphorylation sites of baculovirus-expressed tau and tau constructs. Sf9 cell lysates (72 h after infection) were blotted and incubated with different phosphorylation-dependent and -independent mAbs. The tau constructs are, from left to right: 1) tau23/AP, 2) KXGA/R1, 3) htau23, 4) KXGA/R1/3/4, 5) untransfected Sf9 cell extract (control without tau), 6) Sf9 cells transfected with wild-type baculovirus (control without tau), 7) htau23 expressed in E. coli (unphosphorylated control), and 8) htau23 expressed in E. coli and phosphorylated with brain extract kinase activity (affecting largely the SP and TP sites and inducing a strong M_r shift; see Biernat et al., 1993). Antibodies, from top to bottom: 5E2 (a pan-tau antibody) recognizes all preparations that contain tau. Tau-1 shows only a moderate reaction with tau constructs expressed in Sf9 cells, because a single P site in the vicinity of residue 200 suffices to reduce the binding, but there is a strong reaction with unphosphorylated tau expressed in E. coli or tau23/AP. The antibodies AT-8 to SMI-34 are specific for different phosphorylated SP and TP motifs and therefore show no reaction with the tau23/AP mutant or with E. coli–expressed htau23. Antibody 12E8 recognizes only those constructs that contain Ser262 and/or Ser356 in the repeats. AT-100 recognizes tau phosphorylated at Thr212 and Ser214; this reaction is highly specific for Alzheimer tau but occurs in Sf9 cells as well, provided both sites are phosphorylatable (e.g., not in the tau23/AP mutant).