44-734G

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Peptide Competition. Human recombinant Tau untreated (lane 1) or treated with GSK-3β (1 µg per µg Tau) for 45 minutes (lanes 2-5) was added to background extracts, resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, and then incubated with the Tau (pS199) antibody in a 3% BSA-TBST buffer for two hours at room temperature, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to Tau (pS199) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of Tau (pS199) phosphorylation by the addition of GSK-3β.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Peptide Competition. Human recombinant Tau untreated (lane 1) or treated with GSK-3β (1 µg per µg Tau) for 45 minutes (lanes 2-5) was added to background extracts, resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, and then incubated with the Tau (pS199) antibody in a 3% BSA-TBST buffer for two hours at room temperature, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to Tau (pS199) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of Tau (pS199) phosphorylation by the addition of GSK-3β.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Phospho-Tau (pS199) showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Tau (pS199) polyclonal antibody (Product # 44-734G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Phospho-Tau (pS199) showing staining in the cytoplasm of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Tau (pS199) polyclonal antibody (Product # 44-734G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 10 Biochemical analysis of protein Tau in GSK3alpha deficient mice. A . Biochemical analysis by western blotting of total protein extracts from forebrain of AAC and AA- mice aged 3, 6 and 18 months (n=5/age). Tau protein levels are normalized to actin and expressed relative to control AA- mice at age 3 months. Data (mean+-SEM) are statistically analyzed by two-way Anova (Bonferroni post hoc test), genotype: F (1,24) =3.40, p=0.5093; age: F (2,24) =37.60, p=0.0027; interaction: F (2,24) =0.20, p=0.7759. Lower panels show representative western blots. B . Biochemical analysis by western blotting for phospho-epitopes pS396/404, pS199 and pT231 of endogenous mouse Tau in total protein extracts from hippocampus and forebrain of AAC and control AA- mice. C . Biochemical analysis by western blotting for phospho-epitopes pS396/404, pS199 and pT231 of endogenous mouse Tau in total protein extracts from hippocampus and cortex of GSK3alpha.KO and FVB wild-type mice. D . Biochemical analysis by western blotting for phospho-epitopes pS396/404, pS199 and pT231 of human Tau.P301L in total protein extracts from hippocampus and cortex of GSK3alpha.KOxTau.P301L mice and the parental Tau.P301L mice. In panels B-D , all data are normalized for total Tau and reported relative to the respective control mice. Data (mean+-SEM) are statistically analyzed by unpaired Student's t-test (two-tailed), n=6 or 7 per genotype; * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Tau phosphorylation precludes the BIN1-Tau interaction in vitro . a . Tau-F5 fragment phosphorylation analysis after in vitro phosphorylation by CDK2/CycA3 kinase. Representative immunoblots using various antibodies against phosphorylation epitopes in Tau. The total amount of Tau-F5 was revealed by the phosphorylation-independent antibody Tau5. The significant shift in molecular weight observed in CDK2/CycA3-treated samples indicates Tau hyperphosphorylation. In contrast to other antibodies, the Tau-1 antibody binds to various non-phosphorylated Tau sites; the signal thus decreases when Tau is hyperphosphorylated [ 31 ]. b . 2D [ 1 H, 15 N] HSQC spectra of 125 muM 15 N CDK-phosphorylated Tau-F5[165-245] free in solution (gray) and with a 1 molar amount of GST-BIN1/SH3 (red, superimposed): Overlaid details of 2D [ 1 H, 15 N] HSQC spectra presented in Additional file 6 . c . 2D [ 1 H, 15 N] HSQC spectra of 60 muM 15 N CDK-phosphorylated 2N4R Tau free in solution (gray) and with a 2 molar amount of GST-BIN1/SH3 (blue, superimposed): Overlaid details of full spectra presented in Additional file 7

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Screen for additional pJNK-Tau sites in striatum. (A) Phosphorylation of Tau protein at epitopes subject to phosphorylation by JNK was analyzed by immunoblot. (B) Band optical density (OD) from phosphorylation-specific probes relative to total Tau expression is presented as percent of 2 month-old WT (mean +- SEM) and was analyzed by two-way ANOVA with Bonferroni post-hoc tests comparing each A53T group to age-matched controls (*p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Neuronal uptake of PBS-soluble HMW tau derived from human AD brain. ( a , b ) Primary neurons were incubated with AD or control brain extracts (cases were matched for age and postmortem interval ( Supplementary Table S1 )) and immunostained at day 2 ( a ). ( b ) Quantification of fluorescence intensity of human tau staining. One-way ANOVA and a subsequent Tukey-Kramer test. ( c , d ) Tau uptake ( c ) and seeding activity ( d ) assay in HEK-tau-biosensor cells. (Mann-Whitney U -test) ( e ) Subcellular localization of human tau taken up by neurons (PBS-3,000 g , 500 ng ml -1 human tau). ( f ) Neuron-to-neuron transfer of tau in a 3-chamber microfluidic device. AD brain extract (PBS-3,000 g , 500 ng ml -1 human tau) was added to the 1st chamber. Human tau positive neurons were detected in both the 1st and 2nd chamber at day 7 (arrow). ( g , h ) Quantification of total-tau ( g ) and phospho-tau ( h ) levels in AD and control brain extract (ELISA). Unpaired t -test. ( i ) Brain extracts were immunoblotted with phospho-tau specific antibodies recognizing different epitopes. Representative immunoblot and quantification of phospho-tau levels at each epitope. Unpaired t -test. ( j , k ) SEC analysis of PBS-soluble tau from AD and control brain. ( j ) Representative graph of total tau levels (ELISA) in SEC-separated samples. Small peaks for HMW fractions were detected in both groups (right panel). ( k ) Mean total tau levels of HMW SEC fractions. ( l ) Tau uptake from each SEC

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig.3 Treatment with MO mitigated tau phosphorylation induced by HHcy. A) Levels of tau phosphorylation were measured by western blotting in the hippocampus after treatment (see method). B-E) Quantitative analysis of the blots of phosphorylated tau probed with several phosphorylated-tau antibodies, and (F) non-phosphorylated tau probed with Tau1 antibody, all normalized with total tau (Tau5) level. G) Total tau was normalized to beta-actin. The significant increase in tau phosphorylation at several studied sites was decreased by MO administration. The data were expressed as mean+-SD ( n = 6). # p < 0.05, # # p < 0.01 versus control; * p < 0.05, ** p < 0.01 versus Hcy.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Lack of PBS-soluble phosphorylated HMW tau species is associated with low tau uptake in primary neurons. ( a , top) Uptake of human tau from brain extracts from rTg4510 and rTg21221 mice by primary neurons (PBS-3,000 g , 500 ng ml -1 human tau). Neurons were immunostained with human tau-specific antibody (green) and total (human and mouse) tau antibody (red). ( a , bottom) Tau uptake assay in HEK-tau-biosensor cells. Brain extracts (10 mug protein) were applied to the cells (lipofectamie (-)). ( n =4) Unpaired t -test. Scale bar, 50 mum. ( b ) Human tau levels in brain extracts (ELISA). ( c ) Immunoblot analysis of PBS-soluble extracts with total tau antibody (DA9). Up-shifted bands in rTg4510 brain suggest phosphorylation of tau (arrow). ( d ) Brain extracts were immunoblotted with phospho-tau specific antibodies recognizing different epitopes. Representative immunoblot and quantification of phospho-tau levels at each epitope. ( n =3-4) Unpaired t -test. ( e , f ) SEC analysis of PBS-soluble tau. ( e ) Representative graph of human tau levels (ELISA) in SEC-separated samples ( f ) Mean human tau levels of HMW (Frc. 2-4) and LMW (Frc. 13-16) SEC fractions. ( n =3-6) Unpaired t -test. ( g ) Immunoblot analysis (SDS-PAGE) of SEC-separated fractions from brain extracts (total tau, DAKO). Quantification of band density is also shown (right graphs) ( n =4). Unpaired t -test. ( h ) Dot blot analysis of PBS-soluble brain extracts with tau oligomer-specific antibody (T22), h

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Rapamycin treatment may promote cell morphology and alleviate the Tau phosphorylation of neurons from SAMP8. (A) Cell morphology (magnification, x200). The neurons derived from SAMR1 appear to be in a normal state (a, Neurons-SAMR1). The neurons extracted from SAMP8 were in poor state with most processes broken and exhibited 'bead-like' changes (indicated by the white arrows) (b, Neurons-SAMP8). When pretreated with 0.5 uM rapamycin, neurons from SAMP8 were partially improved with some smooth and slender processes (indicated by the white arrows; c, Neurons-SAMP8 + 0.5 uM rapamycin). When pretreated with 1.0 uM rapamycin, the cell state was worse than the untreated group and most neurons lacked projections (d, Neurons-SAMP8 + 1.0 uM rapamycin). (B) Western blot analysis was used to investigate Tau phosphorylation. In the neurons-SAMP8 group, the protein expression levels of Tau (pS199) and Tau (pS396) were significantly increased when compared with the control neurons-SAMR1 group. When pretreated with 0.5 uM rapamycin for three days, Tau (pS199) and Tau (pS396) exhibited significantly decreased levels of protein expression when compared with the neurons-SAMP8 group. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Treatment with TG mitigated Hcy-induced tau hyperphosphorylation. ( A ) Phosphorylation status of tau protein as measured by Western blotting in the hippocampus after Hcy treatment and TG supplementation. ( B ) Total tau (Tau5) normalized to beta-actin. ( C-E ) Quantitative analysis of the blots of phosphorylated tau probed with several phosphorylated-tau antibodies normalized to total tau (Tau5). A significant decrease in tau hyperphosphorylation at several studied sites was seen following TG administration. The data were expressed as mean +- SEM (n = 6). ## P < 0.01, ### P