MG132 and cycloheximide were purchased from Sigma. Phosphatase inhibitor (PhosSTOP™) and protease inhibitor cocktails (cOmplete™, Mini, EDTA-free) were obtained from Roche. Primary antibodies used in this study included antibodies directed against human Tau (Dako), Otub1, Ubiquitin-Lys48-specific, Ubiquitin-Lys63-specific (Abcam), Phospho-Tau (Ser202/Thr205) AT8, Phospho-Tau (Thr231) AT180, Phospho-Tau (Thr212/Ser214) AT100 (Thermo Fisher Scientific), and Oligomeric-Tau T22 (Merck), applied in combination with appropriate horseradish peroxidase (HRP) or Alexa Fluor® (488/568/647) coupled secondary antibodies.
Phospho tau ser202 thr205 at8
Manufactured by Thermo Fisher Scientific
The Phospho-Tau Ser202/Thr205 (AT8) is a laboratory reagent used for the detection and quantification of phosphorylated tau protein at serine 202 and threonine 205 residues. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Tyrosine hydroxylase, by Merck Group (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
β tubulin, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Mitofusin 2 d2d10, by Cell Signaling Technology (2 mentions)
Drp1 d6c7, by Cell Signaling Technology (2 mentions)
Disposable pellet mixers with pestle motor, by Avantor (2 mentions)
Dopamine d2 receptor, by Merck Group (2 mentions)
Tom20 d8t4n, by Cell Signaling Technology (2 mentions)
Ha tag c29f4, by Cell Signaling Technology (2 mentions)
Complete mini, by Roche (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Oligomeric tau t22, by Merck Group (1 mentions)
α synuclein, by BD (1 mentions)
Ecl substrate, by GE Healthcare (1 mentions)
Gapdh h 12, by Santa Cruz Biotechnology (1 mentions)
4 protocols using phospho tau ser202 thr205 at8
1
Immunoblotting Analysis of Tau Modifications
2
Protein Extraction and Western Blot Analysis of Neural Tissues
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Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0 °C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500 μl, 5 μl aprotinin, PMSF 50 μl, Na3VO4 100 μl and NaF 20 μl in 4.32 ml PBS. Extracts were clarified by centrifugation at 4 °C (13,200g for 20 min). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE [45 (link)]. The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), α-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
3
Quantification of Amyloid-Beta and Tau Proteins
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Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12–15% gels and transferred to a polyvinylidene difluoride membrane. Membranes were then incubated with the following primary antibodies: β-Amyloid (1–42 Specific, D9A3A, Cell Signaling Technology, Danvers, MA, USA, Cat. No. 14974S), Tau (HT7, Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. MN1000), Phospho-Tau (Ser202, Thr205, AT8, Thermo Fisher Scientific, Cat. No. MN1020), GAPDH (H-12, Santa Cruz Biotechnology, Cat. No. sc-166574), and IFITM3 (F-41, Santa Cruz Biotechnology, Cat. No. sc-100768). Peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Sigma-Aldrich, Cat. No. A0545 and A2554) for ECL substrate (GE Healthcare, Barrington, IL, USA) were then employed. To check the quality of p-tau bands, we used CIP at 10 and 40 units (New England Biolabs, Ipswich, MA, USA, Cat. No. M0290) for 60 min at 37 °C. It is important to note that the original blots were cut before hybridization with antibodies. For this reason, full-length blots cannot be provided. The images in Supplementary Fig. S9 depict the blots after cropping.
4
Protein Extraction and Analysis from Brain Tissues
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Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0°C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500μl, 5μl aprotinin, PMSF 50μl, Na 3 VO 4 100μl and NaF 20μl in 4.32 ml PBS. Extracts were clari ed by centrifugation at 4°C (13,200 g for 20 minutes). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE (43) . The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), a-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies.
Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
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