Generated inducible trangenic mice overexpressing p25 in the postnatal forebrain. We used the tetracycline-controlled transactivator (tTA) system to generate bitransgenic mice that inducibly overexpress human p25 under the control of the CamKII promoter.
For immunoblot the following antibodies (diluted 1:1000 unless otherwise noted) were used: monoclonal DC17 (Cdk5) (1:10), polyclonal antibodies p35, p39, P-Nudel (S231), P-mDab1 (S491), P-PSD95 (S19/S25), and P-APP (T668) generated in the Tsai lab; actin and GFAP from Sigma; PSD95, cleaved caspase-3 Asp175 (1:500), p42/44, P-p42/44 (T202/Y204), and P-GSK-3ß (S9) from Cell Signaling Technology; FAK, JNK1, and P-JNK (T183/Y185) from Santa Cruz Technology; P-GSK-3a/ß (Y279/216) and tau 5 from Biosource International (Camarillo, CA); ß-catenin from Becton, Dickinson, and Company (Franklin Lakes, NJ); tau1 (1:2000) from Chemicon International (Temecula, CA); AT8 from Innogenetics (Belgium); and PHF-1 from P. Davies.
The following antibodies were used for immunostaining: GFP (1:1000) and HuC/D (10 µg/ml) from Molecular Probes (Eugene, OR); NeuN (1:500) from Chemicon International; GFAP (1:500) from Sigma; cleaved caspase-3 Asp175 (1:50) from Cell Signaling Technology; AT8 (1:100) and anti-tau AT180 (1:100) from Innogenetics (Belgium); and PHF-1 (1:100) from P. Davies
The following antibodies were used for immunogold labeling. Phosphorylation-independent tau antibodies (1:5–1:10) include tau 5 from Biosource International (Camarillo, CA) and 5E2 from K. Kosik. Phosphorylation-dependent tau antibodies include monoclonal antibodies AT8 from Innogenetics (Belgium) and PHF-1 and TG3 from P. Davies.