Figure 7.
Partition of GSK3 in different samples derived from fractionated tissue extracts of non-transgenic, JNPL3, and TAPP mice. A: Unfractionated (Total) and fractionated samples (SN1-SN4, P2, P4) from spinal cords of three 9-month-old JNPL3 and 9-month-old non-transgenic (NT) mice were immunoblotted with anti-pY279/216 antibody. The sample loading was adjusted as indicated. SN2 fraction prepared from an AD brain (the first lane on each blot) was used as reference. GSK3 immunoreactivity was detected mainly in three bands of molecular weight consistent with GSK3α, and two isoforms of GSK3β (exon 8A+ and exon 8A-). Although JNPL3 samples tended to contain less GSK3 than non-transgenic controls, the differences were not statistically significant. Most GSK3 was recovered in the supernatant fraction (SN1) derived from high-speed centrifugation. PY279/216 immunoreactivity was also detected weakly in proteins of molecular weight about 64 kd (arrow) in the P2 (sarkosyl-insoluble) fraction. The band was not recognized by antibody TPK1/GSK3β PY216 (data not shown). B: Proportion of Y216-phosphorylated GSK3β in different fractions from spinal cords. Percentage of the mean was estimated after densitometric analysis of GSK3β immunoreactive band (46 and 47 kd in molecular weight). Standard errors were shown in parenthesis. C: Partition of GSK3 and phosphorylated tau in different regions of brains from JNPL3 and TAPP mice. Western blotting of SN1 fraction showed no differences between two JNPL3 (BL3, 9 months of age; BJ10, 9 months of age) and two TAPP (BZ6, 6 months of age; BJ2, 9 months of age) mice in the level of 279/216 phosphorylated GSK3α/β (a), inactive form of GSK3β (TPK1/GSK3β-PS9) (b), and total GSK3α/β (c) in the cortico-limbic (1) and subcortical (2) brain regions. Immunoblotting of the P2 fraction demonstrated the labeling of a band ∼64 kd (arrow) by anti-pY279/216 antibody. This band was detected (d) mainly in the subcortical region in JNPL3 mice, and in both the cortico-limbic and subcortical regions in TAPP mice. Immunoblotting with CP13 showed (e) the labeling of 64 kd phosphorylated tau (tau 64 kd). In TAPP mice, the relative level of 64- kd protein in two brain regions detected by GSK3 antibody is higher than by phospho-tau antibody, suggesting that the GSK3 cross-reactive protein is probably not tau.