Cells were harvested and lysed by sonication and boiling in Laemmli buffer. Samples were separated electrophoretically on an SDS gel, transferred onto nitrocellulose membranes and immunoblotted using antibodies for total tau (tau12 antibody provided by Skip Binder, Michigan State), pTau Thr205, pTau Ser262, pTau Ser396 and pTau Ser404 (Invitrogen). The optical density of the bands was quantified using Image J software (http://rsbweb.nih.gov/ij ) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each blot contained 2-4 samples per treatment condition and densitometric analysis was performed on 4-5 separate blots per experiment.
Ptau ser396
Manufactured by Thermo Fisher Scientific
Sourced in Canada
The PTau Ser396 is a laboratory equipment product. It is used to measure the levels of phosphorylated tau protein at the serine 396 site. This measurement can provide information about the biochemical state of the sample being analyzed.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
α tubulin, by Merck Group (1 mentions)
At180, by Fujirebio (1 mentions)
Aβ1 42, by Thermo Fisher Scientific (1 mentions)
Trem2, by Abcam (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Aβ1 40, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Abcam (1 mentions)
Tdp 43, by Proteintech (1 mentions)
Neurofilament light, by UmanDiagnostics (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Anti ubiquitin clone ubi 1, by Merck Group (1 mentions)
Cul4a, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
4 protocols using ptau ser396
1
Quantification of Tau Phosphorylation
2
Antibody Panel for Tau Pathology
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The following anti-tau antibodies were used: HT7 (human-specific Tau; Invitrogen, #MN-1000), Tau-5 (total tau; Biosource, #AHB0042), Tau-12 (total tau; generously provided by Dr. Lester Binder, Northwestern University, IL), TG5 and DA9 (total tau; generously provided by Dr. Peter Davies, Albert Einstein College of Medicine, NY), p-Tau Ser396 (Invitrogen, #44-752G), PHF-1 (p-Ser396/404; Davies), CP13 (p-S202; Davies), AT100 (p-Ser212/214; Pierce, #P10636), AT180 (pThr231/Ser235; Innogenetics, #90337), 12E8 (p-Ser262/356; generously provided by Dr. Peter Seubert, Elan Pharmaceuticals), Tau-1 (dephosphorylated Ser195/198/199/202; Chemicon, #MAB3420), MC-1 (conformation dependent antibody; Davies), LC3 (Novus Biologicals, #NB600–1384), SQSTM1 (Abnova, #H00008878-M01), pALK (p-Tyr1604; Cell signaling, #3341), ALK1 (BD Pharmingen, #559254), ALK (Invitrogen, #51–3900), ALK (DAKO, #IS64130–2), α-tubulin (Sigma, #T5168), β-actin (Sigma, #A2668), and GFP (Santa Cruz, #sc-8334). Anti-ALK monoclonal antibodies, mAb46, mAb30, and mAb13, generously provided from Dr. Marc Vigny (INSERM U440, Paris) [31 (link)].
3
Biomarker Validation in Rhesus Monkeys
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Validation of human ELISAs using rhesus monkey samples were validated in our previous study7 . Aβ1‐42, Aβ1‐40, t‐tau, p‐tau Ser199, p‐tau Thr231, p‐tau Ser396 (Invitrogen), TNF‐α, IL‐6, TREM2, BDNF (Abcam), TDP‐43 (Proteintech), and neurofilament‐light (Uman Diagnostics) ELISA assays were performed according to each kit manufacturer's instructions, after sample dilution optimization.
4
Comprehensive Western Blot and Immunostaining Protocol
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Total tau TAU5 for western blotting (Invitrogen, Rockford, IL), total tau TAU5 for IP (AbCam, Cambridge, MA), total tau K9JA for IF (Dako/Agilent, Santa Clara, CA), P-tau Ser396 (Invitrogen), P-tau PHF-1 (kindly provided by Dr. Peter Davies, Albert Einstein College of Medicine, NY). Neuronal marker MAP2 (Chemicon/Millipore). CRL4CRBN E3 Ligase components DDB1 (AbCam), CUL4A (Cell Signaling Technology), and CRBN (ProteinTech, Rosemont, IL). Anti-Ubiquitin, clone Ubi-1 (Millipore, Darmstadt, Germany). Internal controls GAPDH (Abcam) and β-Actin (Sigma). Nuclear stain Hoechst 33342 (Invitrogen).
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