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. 2006 Jun;26(11):4327–4338. doi: 10.1128/MCB.02393-05

FIG. 3.

FIG. 3.

The AICD induces GSK-3β expression and tau phosphorylation in NGF-differentiated PC12 cells and rat primary cortical neurons, whereas the T668A point mutant does not. (a) For transactivation by APP-Gal4, cells were cotransfected with one of the following: (i) pG5E1B-luc (Gal4 reporter plasmid); (ii) pMst (Gal4), pMst-APP (APP-Gal4), pMst-T668A APP (APPT668A-Gal4), pMst-AICD (AICD-Gal4), and pMst-T668A AICD (AICDT668A-Gal4); or (iii) pcDNA3-Fe65 (Fe65). Analyses were performed as described previously (8). The normalized luciferase activity is expressed as the relative induction over transcription by Gal4 alone. (b) To confirm the interactions between AICDs, Fe65, and CP2, we performed immunoprecipitation and immunoblotting. After immunoprecipitation with each indicated antibody (anti-GFP, anti-flag, or anti-pAPPT668 antibody), immunoblotting was performed using the nuclear fraction obtained from differentiated PC12 cells cotransfected with ACIDs and Fe65 using antibodies against Fe65 or CP2. The expression levels of AICDs in the pEGFP-N1 vector and Fe65 in differentiated PC12 cells were checked using anti-GFP or anti-flag antibodies. (c) At 48 h posttransfection, the changes in GSK-3β protein and p-tau were checked by Western blotting with anti-GSK-3β or AT8 antibody, which recognizes phosphorylated tau at its Ser202 and Thr205 sites, in PC12 cells and rat primary cortical neurons cotransfected with AICDs and Fe65. The level of total tau protein was checked as well as that of β-tubulin. The expression levels of AICDs in pEGFP-N1 vector and Fe65 in differentiated PC12 cells were checked using anti-GFP antibody or anti-Fe65 antiserum. (d) GSK-3β promoter activity was measured by luciferase activity assay in PC12 cells cotransfected with AICDs and human GSK-3β promoter in PGL2 vector. For cotransfection experiments, 4 μg of human GSK-3β promoter in PGL2 vector and AICDs (C50, dC50, or C50 T668A) in pEGFP-N1 vector were added to dPC12 cells. Luciferase activity was measured using a Biocounter M1500 luminometer (Lumac, GE, Groningen, The Netherlands). Protein concentrations were determined using Bradford protein assay reagent (Bio-Rad), and luciferase activities were normalized versus the obtained protein concentrations.