Journal:

Article Title: Presenilin 1 associates with glycogen synthase kinase-3? and its substrate tau

doi:

Figure Lengend Snippet: Correlation between levels of PS1, tau, GSK-3β, and MAPK in human brains of various ages. The Triton X-100 soluble fraction of brain homogenate (60 μg total protein/lane) was examined by Western analysis using anti-PS1 antibody (MKAD3.4) (1:5), anti-tau antibody (JM) (1:5,000), anti-GSK-3β (Transduction Laboratories, Lexington, KY) (1:1,000), anti-MAPK (Santa Cruz) (1:2,000), and anti-NSE (Dako) (1:500). (A) Western blot using the mAb MKAD3.4. Top of each lane shows age of each person. MKAD3.4 reacts with a faint 47 kDa full-length PS1 (FL) and 28–35 kDa N-terminal fragments (NTF). The reactive band was quantified with a Densitograph Lumino-CCD (ATTO Corporation, Tokyo, Japan). The levels of PS1, tau, GSK-3β, and MAPK were normalized to the level of NSE in each sample, and the levels of tau (B), GSK-3β (C), and MAPK(D) were plotted against the corresponding PS1 levels. There were significant correlations between both PS1 and tau (n = 23, slope of regression line = 41.8, R2 = 0.65, P < 0.0001), as well as GSK-3β (n = 23, slope of regression line = 110.9, R2 = 0.61, P < 0.0001). In contrast, a linear regression showed no significant correlation between PS1 and MAPK (n = 23, slope of regression line = −0.2, R2 = 0.04, P > 0.1).

Article Snippet: The reactive band was quantified with a Densitograph Lumino-CCD (ATTO Corporation, Tokyo, Japan).

Techniques: Western Blot, Transduction

Journal:

Article Title: Presenilin 1 associates with glycogen synthase kinase-3? and its substrate tau

doi:

Figure Lengend Snippet: Effect of PS1 mutants on tau phosphorylation. (A) Constructs coding for FAD-associated mutant forms of PS1 was transfected into COS-7 cells. Ten percent of each lysate was used for immunoblotting with MKAD3.4, and the remainder of each lysate was used for analyzing the coimmunoprecipitation of GSK-3β with PS1, using MKAD3.4. Constructs coding for four repeat tau and FAD-associated mutant forms of PS1 were cotransfected into COS-7 cells. Each lysate was then immunoprecipitated by using MKAD3.4 and immunoblotted by using anti-tau antibody (JM). Recovery of GSK-3β (closed bar) and tau (striped bar) is presented as the percentage (%) of recovery normalized to the corresponding expression levels of PS1 (mean ± SD; n = 3). The lower percentage recovery/PS1 on tau is because of underestimation of the cotransfection results. ∗∗, P < 0.005 (two-tailed t test). (B) Constructs coding for four repeat tau and FAD-associated mutant forms of PS1 were cotransfected into COS-7 cells. Each lysate (30 μg) was then immunoblotted by using the phosphorylation-dependent antibody (PS199). The immunoreactive bands were quantified with a Lumino-CCD Densitograph (Atto). The level of tau phosphorylation is presented as mean ± SD (n = 3), ∗∗, P < 0.005 (two-tailed t test).

Article Snippet: The reactive band was quantified with a Densitograph Lumino-CCD (ATTO Corporation, Tokyo, Japan).

Techniques: Construct, Mutagenesis, Transfection, Western Blot, Immunoprecipitation, Expressing, Cotransfection, Two Tailed Test