The cDNA of the human PP2A Ca mutant L199P was isolated from yeast plasmid YEpDE2.5.12 that carries a 978 bp HindIII/BamHI fragment encoding the HA-PP2ACa-2512 mutant allele.
The HA-PP2ACa-2512 allele contains the t196®c transition mutation encoding a L199P amino acid substitution (numbering is for the untagged PP2ACa). It also encodes a c215®t base change which is a silent mutation.
The 978 bp fragment was subcloned into the neuron-specific murine Thy1.2 expression vector.
Transgenic mice were produced by pronuclear microinjection of B6D2F1 x B6D2F1 embryos.
Founders were identified by PCR analysis of lysates from tail biopsies using oligos O-144 (5’-gttcaccaaggagctggaccag-3’) and O-145 (5’-acaggaagtagtctggggtacg-3’), and an amplification product of 915 bp fragment was obtained. Founder animals were intercrossed with C57BL/6 mice to establish lines.
AT8 (Innogenetics Inc., Temse, Belgium, at a dilution of 1:20)
AT100 (Innogenetics Inc., 1:100)
AT180 (Innogenetics Inc., 1:50)
12E8 (Dr. Peter Seubert, Elan Pharmaceuticals, South San Francisco, 1:100)
TG3 (Dr. Peter Davies, Bronx, New York, 1:20)
PHF1 (Dr. Peter Davies, 1:50)
AD2 (Dr. Chantal Mourton-Gilles, Lille, France, 1:500)
MC1 (Dr. Peter Davies, 1:10)
R145d (Dr. Khalid Iqbal, Staten Island, New York, 1:3000)
S199P (Dr. André Delacourte; Lille, France, 1:100)
For Immunohistochemistry, a commercially available polyclonal anti-ubiquitin antibody (Calbiochem, San Diego, CA at a dilution of 1:100).
For immuno-blotting, the following anti-tau antibodies were used:
phosphorylation-independent monoclonal antibody TAU-5 (Neo Markers Inc., Fremont, CA; 1mg/ml),
phosphorylation-dependent antibodies/antisera AT8 (1:150), AT100 (1:1000), AT180 (1:150), AD2 (1:10.000), pS422 (Biosource Inc., Camarillo, CA 1:500) and S199P (1:5000).
Two monoclonal anti-HA antibodies (Roche, Rotkreuz, Switzerland, IH 1:200, WB 1:500, and Santa Cruz Inc., Santa Cruz, CA, IH1:100, WB 1:500) and a polyclonal anti-HA antibody (Santa Cruz, IH 1:500, WB 1:1000) were used.
The rabbit anti-PP2AC antiserum V598A (Promega Inc., Madison, WI) directed against a C-terminal sequence shared between human and mouse PP2A Ca and Cb was used at a 1:100 dilution for immuno-blot analysis.
For immunofluorescence, secondary antibodies were obtained from Molecular Probes (ALEXA-FLUORTM series, Eugene, OR) and for immunoblotting, HRP-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA) were used.
A g-P32 labeled oligonucleotide hybridizing to exon 7 of the human PP2A Ca gene (5’-gacatgtggctcgcctctac-3’)