Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-4

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p> either a low (25 μM), or optimal (75 μM) concentration of ARA (Panels A and B, respectively). Panels C-F show polymerization reaction products in which mock-phosphorylated tau had been polymerized. In panel C and D, GSK-3β had been omitted from the phosphorylation reaction (- GSK + ATP) and in panel E and F, ATP had been omitted from the phosphorylation reaction (+ GSK – ATP). The 25 μM ARA reactions were diluted five fold, the 75 μM ARA reactions were diluted ten fold prior to grid preparation

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-5

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p>.). Panel C and D show products of phosphorylated tau that was polymerized in the presence of 20 μM ThS (+phos., +ThS). The clusters of filaments were approximately 2 μm across their longest axis. Magnification was 20,000 × (scale bar represents 500 nm). Panels A and C were induced with 25 μM ARA and diluted five fold prior to grid preparation; Panels B and D were induced with 75 μM ARA, and diluted 10 fold

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-2

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p>phorylated tau (filled symbols) showed that phosphorylation altered polymerization kinetics, particularly in the first 30 minutes of the reaction. Panel A shows kinetics over the entire 20-h reaction time. Panel B shows only the first 30 minutes (the section boxed by a dotted line in panel A). Two ratios of ARA inducer:tau protein were compared: a suboptimal ratio (labeled 25 μM, representing the concentration of ARA, diamonds) and an optimal ratio (labeled 75 μM, circles). The tau protein concentration was 2 μM for all kinetic reactions. Changes in ThS fluorescence intensity (y axis) was used to indicate the extent of polymerization and measurements were in arbitrary units (a.u.). Error bars are +/- SEM. Every 40th data point was plotted for ease in interpretation

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-9

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p>anes 1, 4, 7, 10, and 13) or the presence of 0.006 U GSK-3β per pmol tau (lanes 2, 5, 8, 11, 14) or 0.018 U GSK-3β per pmol tau (lanes 3, 6, 9, 12, and 15). A definite band shift in the migration of phosphorylated tau can be detected with increasing time and kinase concentration. B) The amount of γ-P incorporation over time using 0.018 U GSK-3β per pmol tau (open circles, right y-axis) is compared to the SDS-PAGE analysis in Panel A (open squares, left y-axis). Lines are drawn through the points to ease comparison. Data represents a single trial

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-3

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p>hown in Figure 3, were diluted five-fold and prepared for transmission electron microscopy (TEM). Panels A, C and E show representative clusters of filaments at a magnification of 50,000 × (scale bar represents 500 nm). Panels B, D and F show the same clusters at a magnification of 100,000 × (scale bar represents 100 nm). In the higher magnification micrographs, white arrowheads denote non-touching filaments; white asterisks, branching filaments; and black arrowheads, tendril-like fibrils that appear to laterally connect larger adjacent filaments

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-6

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p>red by each cluster (size) (Panel B) were determined and plotted against the calculated density of the cluster. Each cluster is represented separately in Panels A and B. This data was then averaged and summarized in Panel C (area), Panel D (filament length) and Panel E (density). Polymerization products from 25 μM ARA-induced polymerization reactions are presented as white circles and bars; those from 75 μM ARA induction are presented as black circles and bars. Error bars are +/- SEM

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology-1

<p><b>Copyright information:</b></p><p>Taken from "Tau phosphorylation by GSK-3β promotes tangle-like filament morphology"</p><p>http://www.molecularneurodegeneration.com/content/2/1/12</p><p>Molecular Neurodegeneration 2007;2():12-12.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1936422.</p><p></p> blots of GSK-3β phosphorylated tau. Panel A and B show two representative blots. Three repetitions of each phosphorylated tau concentration, ranging from 800 to 1.56 ng, were spotted on a blot, then probed with an anti-phosphorylation specific antibody (See Materials and Methods for details). To estimate non-specific background levels, each blot also included one spot of non-phosphorylated tau at each concentration. Following density analysis, the antibody titrations were plotted (Panels D and E) and 1/2 max concentrations were estimated. Anti-S422 antibody shown in Panel C, and its titration plot in Panel F, did not recognize phosphorylation and served as a negative control