- 44-760G - Invitrogen Antibodies MAPT antibody

Submitted references Sphingolipids and Inositol Phosphates Regulate the Tau Protein Phosphorylation Status in Humanized Yeast.

Randez-Gil F, Bojunga L, Estruch F, Winderickx J, Del Poeta M, Prieto JA
Frontiers in cell and developmental biology 2020;8:592159

Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer's disease brain.

Takeda S, Wegmann S, Cho H, DeVos SL, Commins C, Roe AD, Nicholls SB, Carlson GA, Pitstick R, Nobuhara CK, Costantino I, Frosch MP, Müller DJ, Irimia D, Hyman BT
Nature communications 2015 Oct 13;6:8490

Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.

Petry FR, Pelletier J, Bretteville A, Morin F, Calon F, Hébert SS, Whittington RA, Planel E
PloS one 2014;9(5):e94251

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases.

Yu Y, Run X, Liang Z, Li Y, Liu F, Liu Y, Iqbal K, Grundke-Iqbal I, Gong CX
Journal of neurochemistry 2009 Mar;108(6):1480-94

Pseudophosphorylation of tau protein alters its ability for self-aggregation.

Haase C, Stieler JT, Arendt T, Holzer M
Journal of neurochemistry 2004 Mar;88(6):1509-20

Pseudophosphorylation of tau protein alters its ability for self-aggregation.

Haase C, Stieler JT, Arendt T, Holzer M
Journal of neurochemistry 2004 Mar;88(6):1509-20

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Supportive validation

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Experimental details: Up-regulation and Antibody-Peptide Competition: Peptide Competition: Human recombinant Tau untreated (lane 1) or treated with PKA (36 µg per µg Tau) for 1 hour (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 (pS409) antibody in a 1% 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 phosphopeptide corresponding to Tau (pS409) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of Tau (pS409) phosphorylation by the addition of PKA.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Phospho-Tau (pS409) 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 (pS409) polyclonal antibody (Product # 44-760G) diluted in 3% BSA-PBS at a dilution of 1:20 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: 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 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: 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

44-760G

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