APP Processing and Tyrosine682 Figure 1. Generation of APPYG mice. A, Schematic representation of the construct injected in 129 ES cells, showing site of APP T668A and Y682G mutation on last Exon 16, primer sites, site of Southern Blot probe, LoxP, pgk-dta and pgk-Neo sites. The bottom graphics depict the construct with and without the pgk-Neo cassette that has been removed by means of Cre recombinase. B, The right arm (p1–p2) PCR analysis of six positively targeted ES clones. A 3.2 Kb PCR product digested by a novel restriction site Sma I produce 0.4 Kb and 2.8 Kb fragment. C, Southern Blot showing a shift from the 7.5 Kb of the wild type genome to the 6.0 Kb band of two T668A and four Y682G positively knock in ES clones for the homologous recombination of the mutated allele, due to the insertion of a new BamHI site. doi:10.1371/journal.pone.0015503.g001 Figure 2. Altered APP proteolytic processing in APPYG/YG mouse brain. APP2/2 brain was used as a negative control in each experiment. A, Immunoblot analysis comparing full length APP (WH = whole homogenates), sAPP-a, sAPP-b and tubulin levels between APPwt/wt APPYG/YG mice (n = 3). B, Quantitative analysis of panel A normalized to tubulin (*** = p,0.01). C, WB analysis of APPwt/wt, APPwt/TA, APPTA/TA, APPwt/YG and APPYG/YG brain lysate showing full length APP, sAPP-a and sAPP-b. D, WB analysis of APP CTF’s using a tris-tricine gel, and comparing APPwt/wt with APPTA/TA and APPYG/YG. Five specific species representing C83, C89, C99 and their respective phosphorylated forms can be identified, noting that p-C83 and C89 overlap. Bands at the very top and bottom are non-specific. E, Ab4ELISA comparing APPwt/wt with APPTA/TA (n = 3) and APPwt/wt with APPYG/YG (n = 4). APP2/2 mice were also used to validate specificity of the assay (data not shown). doi:10.1371/journal.pone.0015503.g002 PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e15503