Analysis of Tau phosphorylation in 4RTau cell lines by 2D electrophoresis.

<p>A) Bi-dimensional analysis of <i>WT</i> and Mutated <i>P301S</i> or <i>S305N</i> 4RTau. Differentiated cells treated with tetracycline for 48 hours were subjected to 2D analysis and immunoblotted with Tau Exon10 antibody (4RTau specific). Alignments of immunoblots were performed using unspecific products revealed by secondary antibody. Different molecular weight (MW) levels (levels1–3: L1–L3) of Tau isovariants are represented by short dashed lines and pI indicators (obtained with an internal IEF control) by long and short mixed dashed lines. B) Bi-dimensional analysis of <i>WT</i> and Mutated 4RTau after <i>in vitro</i> Tau dephosphorylation followed by immunoblotting with Tau exon10 antibody. C) Immunoblot analysis of <i>in vitro</i> dephosphorylated <i>WT</i> and mutated (<i>P301S</i> and <i>S305N</i>) 4RTau. Differentiated cells treated with tetracycline for 48 hours were subjected (+) or not (−), to an <i>in vitro</i> dephosphorylation by λ-phosphatase and immunoblotted by phosphorylation dependent antibody. Total amount of Tau proteins loaded is visualized with TauCter 1902 antibody (Total Tau) and β-actin was used as loading control.</p

Analysis of Tau aggregation by electronic microscopy and Gold-immunolabeling.

<p>Lysates from differentiated Mock and 4RTau cell lines, treated with tetracycline for 48 hours, were subjected to immunogold labelling with TauCter 1902 (Total Tau) antibody and revealed by colloidal gold labelled secondary antibody. Images were obtained at high magnification: 20 000×. “Clustering” of Tau immunoreactivity is indicated by black arrows.</p

Analysis of overall Tau phosphorylation in THY-Tau 22 transgenic mice.

<p>Comparison of 2D analysis of human exogenous Tau in 3-weeks old Thy-Tau22 trangenic mice and mutated <i>P301S</i> 4RTau from SY5Y Cells. Alignments of immunoblots were performed using unspecific products revealed by secondary antibody. Different molecular weight (MW) levels (L1–L3) of Tau isovariants are represented by short dashed lines and pI indicators (obtained with an internal IEF control) by long and short mixed dashed lines. Exogenous Tau in mice was probed by an anti-human Tau antibody and exogenous <i>P301S</i> 4RTau in SY5Y cells was visualized by Tau-Exon10 antibody.</p

Antibodies used in immunoblotting studies.

*<p>Blocking: Tris-buffered saline, pH 8, 0.05% Tween 20+5% skim Milk.</p

Analysis of transgenes expression in 4RTau cell lines.

<p>A) Lysates from Mock and 4RTau cells, treated or not (0) with tetracycline for 24 to 48 hours were immunolabeled with TauCter 1902 (Total Tau), and β-actin as loading control. B) Quantification of Tau expression levels in 4RTau cell lines: Ratios of densitometric values of TauCter1902/β-actin immunoreactivities are presented. Ratios are normalized to those obtained from <i>WT</i> 4RTau cells after 48 hours tetracycline (arbitrary value = 1).</p