Figure 3.

Increased activation of GSK3β in PS1 ^-/- and PS1 KI ^M146V fibroblasts and neuronal cells expressing PS1 mutations M146V, I143T, and D9. A, Protein expression analysis of PS1 wild-type (WT), PS1 ^-/- (KO), and PS1 KI ^M146V (KI) fibroblasts. The figure shows samples of two different WT, KO, and KI cultures. Reduced levels of GSK3β-Pser9 (inactive form) were detected in PS1 KO and KI as compared with WT fibroblasts. No changes in the expression levels of total GSK3β, kinesin-I heavy chain (KHC), and tubulin were observed. Note the absence of PS1 in KO fibroblast and similar levels of PS1 expression in WT and KI fibroblasts. B, NT2 neuronal cells were transfected with PS1 WT and PS1 mutant M143T, M146V, and D9 constructs. No significant changes were detected in total GSK3β (GSK3β total) and tubulin levels (data not shown). The histogram represents the amount of GSK3β-Pser9 expressed as a percentage of the level in control cells (transfected with GFP). Values are the mean ± SE; n = 3 independent experiments. *p < 0.02 relative to GFP by Student's t test. Transfection and quantitative Western blot analysis were performed as described in Materials and Methods. C, Expression of PS1 mutations does not affect cell viability. Sister cultures to the ones used for the experiment shown in B were used to assess cell viability. The results showed no changes in cell survival associated with the expression of PS1 mutations. Viability was assessed by using a propidium iodide exclusion assay as described in Materials and Methods. At least 200 cells were scored per culture in triplicate cultures. The histogram represents the mean ± SE.