The WKS44 antibody (Fig. phosphorylation is as important as phosphorylation of other residues in tauopathy. = 0.250, activation time 30.0 ms, minimal signal required 8 105, minimal MS2 signal 4 105. A cycle of one full MS scan followed by three data-dependent MS/MS was repeated throughout the entire elution time (60 min). All MS/MS spectra acquired were analyzed using the Thermo Electron Bioworks Browser, which employs the database search SEQUEST algorithms. The MS/MS spectra were first analyzed using non-redundant protein database, taking into consideration differential mass increase (+79.99) due to phosphorylation at serine, threonine, and tyrosine [2,17]. The MS/MS spectra were re-searched using a subset of the database specific for tau isoforms. Only those identified peptides with XCorr Rabbit polyclonal to beta defensin131 scores higher than 1.5 (+1), 2.0 (+2), or 2.5 (+3) and delta score 0.1 where taken into consideration. The MS/MS spectra for the identified peptides were also manually evaluated. 2.5. Immunocytochemistry The spinal cord and brain tissues were embedded in Cyclofenil Tissue-Tek O.C.T. Compound (Ted Pella, Redding, CA), frozen and sectioned into 10-m-thick slices. Triple fluorescence labeling for WKS44, 4G10, and nuclei was carried out sequentially on the same sections as previously described . Tissue sections were fixed utilizing 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room heat. After treatment with a blocking solution made up of 4% normal goat serum, 0.05% Tween-20 in phosphate-buffered saline (pH 7.5) for 1 h, the sections were incubated overnight at 4 C with WKS44 (1:500) and 4G10 antibodies (1:200) diluted in 4% normal goat serum in PBS. After washing with 0.05% Tween-20 in PBS-T, the bound immunoglobulins were detected with secondary antibodies, Alexa 488 (goat anti-rabbit IgG) and Alexa 594 (goat anti-mouse IgG), at 1:200 dilution (Molecular Probes, Eugene, OR). Nuclei staining utilizing DAPI (4,6-diamidio-2-phenylindole; Molecular Probes, Eugene, OR) was employed to confirm the presence of cells. The labeled cells were visualized by confocal microscopy (Olympus BX50). In addition, control experiments utilizing only secondary antibodies were used to determine non-specific labeling and autofluorescence. No non-specific labeling or autofluorescence was detected in experimental conditions. Sections were examined for fluorescent signals using excitation and barrier filters appropriate for selective visualization of fluorescein isothiocyanate and Texas red. 3. Results 3.1. Immunoprecipitated tau from S1 fraction is usually tyrosine phosphorylated Tau proteins were immunoprecipitated from S1 (soluble) and P3 (sarkosyl-insoluble) fractions from Cyclofenil brain extract of 10-month-old JNPL3 mice utilizing the human tau-specific monoclonal Tau12 antibody (aa 9C18). As revealed by Western blotting analysis using human tau-specific polyclonal E1 antibody (aa 19 C 33), tau proteins were efficiently immunoprecipitated from both S1 and P3 fractions (Fig. 1A, IP). The immunoprecipitated proteins correspond to the tau proteins observed in S1 and P3 fractions obtained from 9-month-old JNPL3 Cyclofenil mouse brain extract (Fig. 1A, compare In and IP). As control, protein A-sepharose beads incubated with S1 and P3 fractions (Fig. 1A, beads control) and Tau12 immunoprecipitates derived from NT littermate mice (Fig. 1C, NT) were used. The Tau12 immunoprecipitates from 9-month-old JNPL3 mice, but not protein A-sepharose beads and NT littermates, contain tau proteins readily detectable with antibody E1 (Figs. 1A and B). Furthermore, analysis of the supernatant after immunoprecipitation (Fig. 1B, Sup) indicates that most tau proteins were efficiently precipitated. These results indicate that Tau12 specifically immunoprecipitates human tau proteins expressed in JNPL3 transgenic mice. Open in a separate windows Fig. 1 Immunoprecipitated tau from JNPL3 mice Cyclofenil is usually tyrosine phosphorylated. (A) Brain samples from JNPL3 (P301L) and non-transgenic (NT) littermate were homogenized and subjected to fractionation as layed out in Materials and methods. The S1 (soluble) and P3 (sarkosyl-insoluble) fractions were subjected to immunoprecipitation experiments, using monoclonal tau-specific antibody Tau12. As control, protein A-sepharose beads (A, beads control) were used to determine the specificity of Tau12 antibodies (T12)..