44-752G

antibody from Invitrogen Antibodies

Targeting: MAPT DDPAC, FLJ31424, FTDP-17, MAPTL, MGC138549, MSTD, MTBT1, MTBT2, PPND, PPP1R103, tau

Provider product page for 44-752G

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [67]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [2]
Other assay [35]

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Product number: 44-752G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-Tau (Ser396) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Tau. The final product is generated by affinity chromatography using a Tau-derived peptide that is phosphorylated at serine 396.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

MAPT protein structure - 44-752G shown in red.

Supportive validation

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Experimental details: Immunofluorescent analysis of Tau pSer396 in human iPSC-derived forebrain organoids derived at Day 40. The organoids were fixed with 4% PFA for 1 hour at room temperature, followed by incubation with 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT and cryosectioned at 5 µm, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were stained with a rabbit Tau pSer396 polyclonal antibody (red; Product # 44-752G) at a dilution of 1:500 in blocking buffer overnight at 4°C, and then incubated with Donkey anti-Rabbit Alexa Fluor 568 (Product # A10042) at a dilution of 1:1000 as well as DAPI (blue; 1:25000) in blocking solution at room temperature for 1 hour. Images were taken at 20X magnification. Data courtesy of Dr. Zhexing Wen at Emory University.

Supportive validation

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Experimental details: Immunofluorescent analysis of p-Tau (S396) and Tau in human iPSC-derived forebrain organoids derived at Day 40. The organoids were fixed with 4% PFA for 1 hour at room temperature, followed by incubation with 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT and cryosectioned at 5 µm, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were stained with a Mouse Tau (TAU-5) monoclonal antibody (green; Product # AHB0042) at a dilution of 1:500 and a Rabbit p-Tau (Ser396) polyclonal antibody (red; Product # 44-752G) at a dilution of 1:500 in blocking buffer overnight at 4°C, and then incubated with Donkey anti-Mouse Alexa Fluor 488 (Product # R37114), Donkey anti-Rabbit Alexa Fluor 568 (Product # A10042) at a dilution of 1:1000 as well as DAPI (blue; 1:25000) in blocking solution at room temperature for 1 hour. Images were taken at 20X magnification. Scale bar: 50 µm. Data courtesy of Dr. Zhexing Wen at Emory University.

Supportive validation

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Supportive validation

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Experimental details: Figure 7 Analysis of tau signal with polyclonal antibodies by Western blotting. Proteins were extracted from the cortex of 3 mouse lines: control mice (WT and Hypothermic), Tau KO mice and 3xTg-AD mice. Proteins were separated by SDS-PAGE and then identified with the following polyclonal antibodies: A: Total Tau, B: pS199, C: pS396, D: pS404, E: pT205, F: pS422, G: pS262 and H: pS409. Normal anti-rabbit secondary antibodies were used to detect primary antibodies. The heat stable fraction was used to remove non-specificity: I: pS262 and J: pS409. Quantifications of the blots are available in Figure S5 .

Supportive validation

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Experimental details: Figure 3 Abeta1-42 monomers promote phosphorylation at particular sites that have been related to Alzheimer's disease (AD) progression. Representative Western blot of brain extracts (20 mug protein) from control (saline) and treated (Abeta1-42 peptides by ICV for 3 h) mice using antibodies specific for the detection four pathological Tau phosphorylation sites: AT8, pS396, pS262, and pS422. An antibody raised against GAPDH or Tau 5 served as loading control. Densitometric quantification shows an increase of the total protein level of AT8, pS396, and pS422 induced by monomers while monomeric and oligomeric preparations did not change pS262 expression. The data are mean +- standard error of the mean (SEM), * P < 0.05; *** P < 0.001 vs. control by one-way ANOVA followed by Bonferroni post hoc test, n = 6.

Supportive validation

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Experimental details: Figure 7 Hypoxia or gamma -taxilin ablation induces tau hyperphosphorylation and cleavage in neuronal cells. ( a ) Western blot shows hyperphosphorylation at T231 (upper panel) and S396 (middle panel), and tau protein (~55 kDa) (lower panel) in SK-N-SH neuronal cells cultured under hypoxic conditions for indicated durations. Note that cleaved (~35 kDa) tau contains phosphorylated T231, but not phosphorylated S396. ( b ) Western blot shows hyperphosphorylation of tau protein (T231, S396, and S404) in SH-SY5Y neuronal cells cultured in the presence of gamma -taxilin-specific small interfering RNA (siRNA), but not in alpha NAC siRNA-treated cells. Note that total tau increases in amount after gamma -taxilin-specific siRNA addtion. ( c ) Confocal microscopy shows tau aggregations (NFT) in the cytoplasm of gamma -taxilin-specific siRNA-treated SH-SY5Y cells. Scale bar, 10 mu m. ( d ) Tau cleavage is calpain-dependent. Western blot analysis shows that calpain inhibitors (inh3, calpain inhibitor III; or calp, calpeptin) inhibit tau cleavage, but not tau hyperphosphorylation in SK-N-SH neuronal cells (right panels). As expected, GSK-3 beta inhibition with CHIR inhibits tau hyperphosphorylation in the neuronal cells. ( e ) Downregulation of gamma -taxilin and alpha NAC in the brains of patients with AD. Immunohistochemistry shows gamma -taxilin and alpha NAC protein expression in the healthy brain (control, left panels), but not in the brains from patients with AD (right panels). Uppe

Supportive validation

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Experimental details: Figure 4 Res attenuates FA-induced tau protein hyperphosphorylation in N2a cells. (A) N2a cells were pre-treated or post-treated with 10 muM of Res for 30 min before or after 0.5 mM FA exposure for 4 h, or treated simultaneously with 0.5 mM FA for 4 h. (B) N2a cells were pre-treated with Res (0.1, 1, 2.5, 5, and 10 muM) for 30 min, and then treated with 0.5 mM FA for 4 h. Total and phosphorylated tau protein levels were determined by Western blotting (A,B) . Densitometry values were calculated from independent experiments performed in triplicate ( n = 3) and presented as mean +- SEM. * p < 0.05, *** p < 0.001 vs. control. ## p < 0.01, ### p < 0.001 vs. FA treatment group.

Supportive validation

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Experimental details: Figure 1 Tau is rapidly dephosphorylated site-specifically in mouse brains during postmortem. Mice were scarified by cervical dislocation. The dead animal bodies were kept at room temperature for the indicated periods of time. ( A ) Phosphorylation of tau was analyzed by Western blots developed with phosphorylation-dependent and site-specific tau antibodies indicated at the right side of the blots. ( B ) The levels of tau phosphorylation at individual sites were quantified and normalized with total tau level and presented as mean +- SEM. (n = 3-4). * p < 0.05; ** p < 0.01.

Supportive validation

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Experimental details: Figure 1 Tau acetylation modulates tau phosphorylation at specific epitopes. (a-c ) Immunoblot analysis and quantification using the indicated tau antibodies for (a ) cells expressing WT tau-T40 and either an active form of CBP or a catalytically inactive form of CBP (CBP-LD), ( b ) cells co- expressing tau-T40 (WT, DeltaKK, 4KQ, or 4KR) and tau modifying enzymes, and ( c ) cells expressing tau-T40 (WT, DeltaKK, 2KQ, 4KQ, or 4KR) and treated with control (DMSO) or okadaic acid (OA), where indicated. Solid black arrows highlight the ~75 kDa phospho-tau species that is reduced upon acetylation. Statistical significance was assessed using a student t-test (****p < 0.0001). Cropped images from full size immunoblots are provided in panels a-c. Full-length immunoblots are presented in Supplementary Fig. S1 . ( d ) Immunofluorescence microscopy of primary cortical neurons expressing WT-tau-GFP (left) or 4KQ-tau-GFP (right) detecting phosphorylated tau (AT8). White arrows identify transfected neurons. Scale bar, 50 um.

Supportive validation

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Experimental details: FIGURE 4 Cornel iridoid glycoside (CIG) inhibited tau phosphorylation and accumulation in the brain of P301S transgenic mice. (A) Representative images of immunohistochemical staining of AT8 in the cerebral cortex, and hippocampus CA1, CA3, and DG areas of mice. Scale bar = 50 mum. Quantitative analysis of integrated optical density (IOD) of AT8 in the cerebral cortex (B) and hippocampus CA1 (C) , CA3 (D) , and DG (E) areas of mice. (F) Representative images of Western blotting for tau5 (total tau), T22 (tau oligomers), monomer (low exposure), and oligomers (high exposure) of phosphorylated (p)-tau at Ser396 (pS396) in the brain of mice. Quantification of Western blotting for tau5 (G) , T22 (H) , pS396-tau oligomers [140~170 kDa, (I) ], and monomer [~55 kDa, (J) ]. Quantification of blots after normalization with GAPDH, and the numerical value of nTg group was taken as 1. Data are expressed as mean +- SEM; n = 3 per group for immunohistochemical staining; n = 4 per group for western blotting. ## P < 0.01, ### P < 0.001, P301S transgenic mice vs nTg control mice; * P < 0.05, ** P < 0.01, drug-treated P301S mice vs saline-treated P301S mice.

Supportive validation

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Experimental details: FIGURE 3 Age-related changes in monkey S235 and S396 validate mass spectrometry results. The 19.5-year-old animal denoted as ""low"" in the proteomics study and western blots is indicated by a gray square in the graphs. (A) Bar graph summarizing a means comparison of pS235-tau between young (light blue, N = 3) and aged (dark blue, N = 11) macaques. Individual data points are plotted for each group. Due to a lack of normal distribution of pS235-tau with age the means were compared using a Mann-Whitney test (* p = 0.0385). (B) Quantification of pS235-tau normalized by GAPDH is plotted by age. Samples were not normally distributed so the correlation coefficient was computed using a Spearman correlation ( r = 0.5809, * p = 0.0319) but a standard linear regression is represented on the graph. (C) Immunohistochemistry revealed robust immunolabeling for pS235-tau in aged macaque dlPFC layer III and layer V/VI, particularly in pyramidal neurons along apical dendrites and in the cell body. Scale bar, 50 mum. (D) A bar graph summarizing a means comparison of pS396-tau between young (light pink, N = 3) and aged (red, N = 11) macaques. Individual data points are plotted for each group. Given the normal distribution of pS235-tau with age but unequal variance, the means were compared using a Welch's t -test (** p = 0.0013). (E) Quantification of pS396-tau normalized by GAPDH is plotted by age. Samples were normally distributed so the correlation coefficient was computed using a Pearson cor

Supportive validation

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Experimental details: Fig. 4 ALK regulates tau-mediated neurodegeneration in tauopathy fly models. A Enhancement of the rough eye phenotype and disordered ommatidial morphology in tau transgenic flies expressing active Drosophila Alk (Alk ACT ). External phenotypes of fly eyes were examined under a microscope. Control and experimental genotypes: Control (GMR-GAL4/+), Alk ACT (GMR-GAL4/+; UAS- Alk ACT /+), Alk DN (GMR-GAL4/+; UAS- Alk DN /+), tau (GMR-GAL4/+; gl-tau2.1/+), tau + Alk ACT (GMR-GAL4/+; gl-tau2.1/+; UAS- Alk ACT /+) and tau + Alk DN (GMR-GAL4/+; gl-tau2.1/+; UAS- Alk DN /+). B Exacerbation of retinal degeneration by ALK in tau flies. Internal retinal sections were stained with Hoechst 33342, after which they were observed under a fluorescence microscope (upper). Retinal thickness was quantified ( n = 3) (lower). C Increased tau phosphorylation in tau- and Alk ACT -expressing flies. Heads of the flies expressing tau alone or together with an Alk mutant were examined by western blotting. The signals of total tau and phosphorylated tau on the blots were quantified by densitometric analysis and the values are noted under the blot. D Shortened lifespan of transgenic flies expressing tau and Alk FL in neurons. At least 200 flies of each genotype were collected and assayed for longevity. Control and experimental genotypes: Control (elav-GAL4/+), tau (elav-GAL4/+; UAS-tau), Alk FL (elav-GAL4/+; UAS- Alk FL ) and tau + Alk FL (elav-GAL4/+; UAS-tau; UAS- Alk FL ). E Extended lifespan of transgen

Supportive validation

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Experimental details: Fig. 5 ALK is upregulated in AD brains and pharmacological inhibition of ALK rescues memory impairment in 3xTg-AD mice. A Increased expression of ALK and accumulation of p62 in AD brains. Hippocampal lysates from normal elderly controls and patients with AD were analyzed by western blotting (left). Total ALK levels were quantified and normalized to beta-actin. Bars depict mean +- SEM; unpaired two-tailed Student's t test (right). B Colocalization of ALK and phosphorylated tau within the cortex of patients with AD. C-E LDK378 rescues memory in 3xTg-AD mice. Six-month-old 3xTg-AD mice were intraperitoneally injected with 5 mg/kg LDK378 or DMSO (Vehicle) ( n = 7-9) daily for 4 weeks and then analyzed using Y-maze ( C P = 0.0027), novel object recognition ( D P = 0.0046) and passive avoidance ( E ) tests. F-H LDK378 reduces tau phosphorylation and accumulation in the brains of 3xTg-AD mice. Hippocampal extracts from the mice analyzed in ( C - E ) were examined by western blotting ( F ). Levels of total tau (TG5), exogenous human tau (HT7), phosphorylated tau (PHF-1, CP13), p-ERK, and ERK on the blots were quantified and normalized to beta-actin. Bars depict mean +- SEM. PHF-1, P = 0.0059; CP13, P = 0.0319; HT7, P < 0.0001; one-way ANOVA followed by Tukey's test ( G ). The CA1 region in the hippocampus of 8-month-old 3xTg-AD/Vehicle and 3xTg-AD/LDK378 mice were immunostained with anti-MAP2B and AT100 antibodies (Representative confocal images, 200x, Scale bar, 50 mum) ( H ). I The

Supportive validation

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Experimental details: Fig. 6 Immunoblotting using hTg-Tau and Tau-KO brain lysates confirms the specificity for tau for a panel of antibodies. a ) Scheme of tau (2N4R) showing the epitopes of the Tau12, Tau5, Tau1, HT7, BT2 and Dako antibodies. b ) - d' ) Immunoblots demonstrate strong reactivity of all antibodies with tau bands between 40 and 60 kD. Minor non-specific bands present in Tau-KO brain lysates were detected in d ), e ), i ), h ), p ), x ), y ) and c '). Isoform-specific antibodies in h ), i ), j ) and k ) were validated using a tau ladder with all 6 recombinant human tau isoforms. kDa sizes for the marker are given in e' ). Tg: hTg-Tau mouse, KO: Tau-KO mouse brain lysate, TL: recombinant tau ladder

Supportive validation

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Experimental details: Figure 2 Both HMW-tau and LMW-tau are selectively hyperphosphorylated in AD brain. (A) AD and control human brain homogenates were analyzed by western blots developed with the indicated site-specific and phosphorylation dependent anti-tau antibodies. (B,C) Blots were analyzed by densitometry. The levels of hyperphosphorylated HMW-tau (B) and LMW-tau (C) are shown as scattered dots with mean +- SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Supportive validation

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Experimental details: Fig. 1 Tau phosphorylation increases at multiple sites in the brain following r-mTBI in 3xTg-AD mice. Adult female 3xTg-AD mice, 4-- months of age, were subjected to repetitive mild closed-head brain impacts, totaling five with 48-h inter-impact intervals, and sacrificed 24 h after the final impact. A) Representative western blots showing the level of indicated proteins/phosphorylation sites. B) Densitometric quantification of the blots. Among the phosphorylation sites examined, tau was hyperphosphorylated at pThr 205 , pSer 262 and PHF-1 (pSer 396/404 ) sites. Data are presented as scatter dot plots with mean+-SEM ( n = 6-7 mice each) and analyzed using unpaired t test, with Welch's correction in the case of unequal variance.

Supportive validation

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Experimental details: Fig. 5 Translocated I 2 PP2A /SET is colocalized with hyperphosphorylated tau in the neuronal cytoplasm in cerebral cortex of 3xTg-AD mice with r-mTBI. Brain sections were fluorescently dual immuno-stained for I 2 PP2A and phospho-tau. Translocated I 2 PP2A colocalized with pThr 205 -tau, pSer 396 -tau and with pSer 262 -tau in the cytoplasm of neurons (arrows). However, there was no somatodendritic phospho-tau staining in neurons where cytoplasmic I 2 PP2A was absent (filled arrowheads). Scale bar = 10 mu m for all images.

Supportive validation

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Experimental details: Fig. 2 Compared to Braak 0-I samples, many but not all, tau phosphorylation events are increased in native Braak III-IV samples. Normalized phospho-tau signals obtained from ELISA measurements of samples from a ) entorhinal cortices (EC), b ) hippocampi (Hip) and c ) temporal cortices (TC). Student's t-tests: *, p < 0.05, **, p < 0.01, ***, p < 0.001

Supportive validation

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Experimental details: Figure 2. Concentration effect of QC-01-175 ( A ) on tau protein levels of A152T and control neurons. Analysis of total tau (TAU5) and phospho-tau (S396 P-tau) levels upon treatment by western blot ( B ) and ELISA ( C ). Analysis of total tau (TAU5) and phospho-tau (S396 P-tau) levels upon treatment with the negative control QC-03-075 ( D ), by western blot ( E ) and ELISA ( F ). Representative western blots are shown ( B, E ) with mean densitometry quantification (bands corresponding to brackets)+-SEM ( n = 3). ( C, F ) For ELISA, data points represent mean tau levels (mug of total protein) normalized to vehicle-treated +- SEM ( n = 4). Both assays show QC-01-175 dose-dependent effect on tau levels, with QC-03-075 minimal effect (~10%). ( G ) IF of A152T neurons treated with vehicle or 10 muM compound, immuno-probed for total tau (K9JA, red), P-tau (PHF-1, red) and the neuronal marker MAP2 (green), scale bar 50 mum. ( H-J ) Tau ELISA of control neurons treated with QC-01-175, which did not show a dose-dependent effect. ( H ) 8330-8-RC1 line; ( I ) MGH2069-RC1 line; ( J ) CTR2-L17-RC2 line. Data points represent mean tau levels (mug of total protein) normalized to vehicle-treated +- SEM ( n = 3). All neurons were differentiated for 6 weeks and treated with compound for 24 hr. Student T-test between each concentration and vehicle-treated tau levels ns p> 0.05, *p

Supportive validation

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Experimental details: Figure 3. Mechanism of QC-01-175 clearance of tau is CRL4 CRBN and UPS-dependent. Neurons were pre-treated for 6 hr with ( A ) either CRBN ligand excess lenalidomide or tau ligand excess T807, ( B ) the NAE inhibitor MLN4924, the autophagy inhibitor Baf.A1, or ( C ) the proteasome inhibitor carfilzomib; followed by 18 hr treatment with QC-01-175 (or negative control QC-03-075), for a total of 24 hr. Total (TAU5) and P-tau S396 levels were analyzed by western blotting. ( A-C ) Representative blots are shown. ( D-F ) Densitometry bars represent tau mean intensity values +- SD ( n = 3), relative to vehicle-treated samples. Student T-test of QC-01-175 samples relative to vehicle treated, and the remainder bars show p-value of each pre-treatment relative to QC-01-175 to assess rescue of clearance effect (***p

Supportive validation

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Experimental details: Figure 4. Demonstration of ternary complex formation in A152T neurons upon QC-01-175 treatment, by co-IP and western blot analysis. Neurons (6-week differentiated) were treated for 4 hr with 1 muM QC-01-175 +- 30 min pre-treatment with proteasome inhibitors (carfilzomib or bortezomib at 5 muM), with the goal of capturing maximum molecular interactions at 4 hr and halting tau clearance. QC-03-075 is a negative control for CRBN binding. ( A ) Co-IP by DDB1 pulldown and detection of tau in the complex by probing for total tau (TAU5), P-tau S396/S404 (PHF-1), and ubiquitinated proteins (Ubi-1). ( B ) Co-IP by tau pulldown (TAU5) and detection of CRL4 CRBN subunits CRBN, CUL4A and DDB1. Western blot of total tau (K9JA) was used as a control. ( C ) Control western blot analysis with 3% (10 mug) of IP input confirms the effect of QC-01-175 +- proteasome inhibitors on tau and P-tau S396. Red arrows and brackets indicate the predicted bands for each immunoprobed protein ( n = 3).

Supportive validation

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Experimental details: Figure 5--figure supplement 1. Degrader concentration and time effect on tau, in A152T and P301L neurons. ( A-C ) Concentration and time effect of QC-01-175 on total tau (TAU5) and S396 P-tau levels, in A152T 6-week differentiated neurons, treated for ( A ) a short 4 hr interval, ( B ) an intermediate 8 hr interval, ( C ) compared to 24 hr treatment. ( D ) Concentration effect of QC-01-175 4 hr treatment in A152T neurons, by western blot analysis of total tau (TAU5) and P-tau S396 (representative blot is shown) with mean densitometry quantification (bands corresponding to brackets)+-SEM ( n = 3). ( F-H ) Concentration and time effect of QC-01-175 on total tau (TAU5) and S396 P-tau levels, in P301L 6-week differentiated neurons treated for ( F ) 4 hr, ( G ) 8 hr and ( H ) 24 hr. ( A-C, F-H ) Data points represent mean levels of tau protein measured by ELISA, normalized to total mug of protein and to vehicle-treated +- SEM ( n = 3). Student T-test between each dose and vehicle-treated tau levels ns p>0.05, *p

Supportive validation

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Experimental details: Figure 3 Tau phosphorylation in the ipsilateral cortex in Tg/hTau mice injected with AD P-tau. Phosphorylation of tau at Ser199, Thr205, Ser214, Thr217, Ser262, Ser396, Ser404 and Ser422 in the ipsilateral cortex was analyzed by Western blots (A) and analyzed with unpaired student t -test. Data are presented as scattered dots with mean +- SD (B) ; * p < 0.05.

Supportive validation

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Experimental details: Figure 5 Memantine enhances tau phosphorylation after 28-day chronic restraint stress (CRS) in the mouse frontal cortex. (a) Results of Western blotting showed that memantine enhances tau phosphorylation at the AT8 site for both doses. Furthermore, the expression of the 3R tau isoform (RD3), 4R tau isoform (RD4), P-tau-PS396, and TAU5 was also detected among the four groups of mice. (b) Densitometric quantification of the bands in (a). (c) Representative areas of AT8-positive immunofluorescent staining in cortices from the four groups of mice. (d) Western blotting analyzed the effects of memantine administration with doses of 5 and 10 mg/kg for 28 days on tau phosphorylation and expression in frontal cortex of mice without CRS. (e, f) Densitometric quantification of the bands in (d). The results are shown as means +- SEM ( n = 4 in each group). GAPDH was used as a loading control. * p < 0.05; ** p < 0.01; *** p < 0.001. Con: control.