Western blotting was performed as described previously (Engel et al., 2017 (link)). Following quantification of protein concentration, 30 μg of protein samples were boiled in gel-loading buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and probed with the following primary antibodies: Tau-1 (1:1,000, Merck Millipore, Arklow, Ireland), AT8 (1:100, Invitrogen, CA, USA), PHF-1 (1:1,000, Abcam, Cambridge, UK) and GAPDH (1:1,000, Cell Signalling, Dublin, Ireland). Next, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Isis Limited, Bray, Ireland). Protein bands were visualized using Fujifilm LAS-4000 system (Fujifilm, Tokyo, Japan) with chemiluminescence.
Phf 1
Manufactured by Abcam
Sourced in United States, United Kingdom
PHF-1 is an antibody that recognizes the phosphorylated form of the Tau protein. It is designed for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Tau 1, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Las 4000 system, by Fujifilm (1 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
Anti gfap, by Abcam (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Alexa fluor 680 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Bbs nc 6 h14, by Agilent Technologies (1 mentions)
Ps214, by Abcam (1 mentions)
Ac 15, by Merck Group (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Mouse anti α syn, by BD (1 mentions)
Mouse anti th, by Merck Group (1 mentions)
Miniprotein 3 wet transfer unit, by Bio-Rad (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Il 1β, by Merck Group (1 mentions)
Synaptophysin, by Abcam (1 mentions)
7 protocols using phf 1
1
Quantification of Tau Phosphorylation by Western Blot
2
Antibody Validation for Neurodegeneration
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Akt (pan, cat#4691), phospho-Akt (Ser473, cat#4060), phospho-GSK3β (Ser9, cat#5558) were all rabbit antibodies and purchased from Cell Signaling (Danvers, MA, USA). Anti-GSK3β rat (cat#MAB2506), Legumain/asparaginyl endopeptidase sheep (Cat#AF2058) antibodies were purchased from R&D System (Minneapolis, MN, USA). Anti-GFAP (cat#ab7260), phosphor-Tau (S396, cat#ab109390) and PHF1 (cat#ab66275) rabbit antibodies were purchased from Abcam (Cambridge, UK). Anti-NeuN antibody (cat#SIG-39860) was obtained from Biolegend, San Diego, CA, USA. Anti-IAB1 antibody (cat#PA5–21274), anti-tau AT8 antibody (cat#MN1020) and DAPI (cat#62248) were obtained from Thermo Fisher Scientific, Waltham, MA, USA. All the secondary antibodies and anti-β-actin antibody were bought from Antegene (Wuhan, China). The thermoplastic polyetherketone cast elements, which were used as shear stress modifier, were purchased from Promolding (Den Haag, Netherlands). Anti-Tau, AEP-cleaved (N368) rabbit antibody was from Dr. Keqiang Ye at Emory University.
3
Quantifying PTEN-Tau Co-Localization in Alzheimer's Cells
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APPswe cells were seeded into 20-mm-diameter confocal glass-bottom cell culture dishes at a density of 1.0 × 105 cells and transfected with PTEN-overexpressing plasmid. 48h after transfection, the cells were processed as described previously.57 (link) Fluorescence images and data were acquired using a laser confocal microscope (LSM780; Carl Zeiss; Jena, Germany) with 20×/numeric aperture (NA) = 0.5 and 40×/NA = 0.75 objectives, using primary antibodies against PHF-1 (Abcam, Cambridge, MA, USA), Tau (Proteintech, Chicago, IL, USA), and PTEN (Abcam). Fiji ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to provide a quantitative measurement of the co-localization of PTEN with PHF-1 and Tau by Pearson’s correlation coefficient, which estimated the degree of overlap between fluorescence signals obtained in two channels. The degree of co-localization from the Pearson’s coefficient values was categorized as very strong (0.85 to 1.0), strong (0.49 to 0.84), moderate (0.1 to 0.48), weak (−0.26 to 0.09), and very weak (−1 to −0.27) based on a previously published description.58 (link)
4
Quantitative Western Blot Analysis of Tau Proteins
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Proteins were separated on 10 % (w/v) SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking, membranes were incubated overnight at 4 °C with primary antibodies directed against total tau (rabbit polyclonal, 1/10,000, DAKO; mouse monoclonal CP27, 1/10,000, gift from Peter Davies), GST (goat polyclonal, 1/2000 GE Healthcare), phosphorylated tau (mouse monoclonal PHF-1, 1/5000, and CP13, 1/400, both gifts from Peter Davies; rabbit polyclonal pS214, 1/500, Abcam; rabbit polyclonal pS262, 1/500, Abcam), dephosphorylated tau (mouse monoclonal Tau-1, 1/5000, Millipore), fyn (rabbit polyclonal, 1/1000, HPA023887, Sigma), β-actin (mouse monoclonal, AC15, 1/10,000, Sigma), and neuron-specific enolase (mouse monoclonal, BBS/NC/VI-H14, 1/10,000, DAKO). Blotted membranes were incubated with secondary antibodies (Alexa Fluor® 680 goat anti-mouse, 1/10,000, Life Technologies or IRDye™ 800 conjugated goat anti-rabbit, 1/10,000, Rockland Immunochemicals Inc.) and antigens were visualised and quantified using an Odyssey scanner (Li-Cor Biosciences).
5
Protein Expression Analysis in Brain Samples
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The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h [24 (link)]. Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit anti-phosphorylated Tau Ser396/Ser404 (PHF-1, 1:1000, Abcam, ab184951) then followed by overnight incubation at 4 °C. The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, and signals were detected by chemiluminescence (ECL, Pierce, USA). Images were acquired with Image Quant LAS 4000 mini (GE Healthcare, USA) and analyzed using Image J. Analyses were completed in duplicate and the mean value was calculated.
6
Western Blot Analysis of Neurodegeneration
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Samples in soluble fraction were loaded into a 4–20% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad), and transferred to PVDF Immobilon FL membranes (Millipore). After blocking with 5% non-fat milk in PBS, membranes were blotted overnight with primary antibodies in 5% non-fat milk containing 0.01% Tween-20, and then probed with LI-COR IRDye secondary antibodies or horseradish peroxidase-conjugated secondary antibody, detected by SuperSignal West Femto Chemiluminescent Substrate (Pierce). The information of primary antibodies and their dilutions used in this study are as follows: Cleaved Caspase-3 (Cell Signaling Technology, 9661, 1:1000), Caspase-3 (Cell Signaling Technology, 9662, 1:1000), PSD95 (Abcam, ab2723, 1:1000), Synaptophysin (Abcam, ab8049, 1:1000), AT8 (Thermo Fisher Scientific, MN1020, 1:1000), PHF1 (Abcam, ab184951, 1:1000), Tau5 (Millipore, 577801, 1:1000), APP (Thermo Fisher Scientific, 14-9749-82, 1:1000), GFAP (Millipore, MAB360, 1:1000), EEA1 (Cell Signaling Technology, 2411, 1:1000), LAMP1 (Cell Signaling Technology, 9091, 1:1000), G3BP (BD, 611126, 1:1000), ERCC4 (Fitzgerald, 10R-4026, 1:1000), POLR3A (Abcam, ab96328, 1:1000), and HSPA4 (Cell Signaling Technology, 3303, 1:1000). All uncropped blot images with molecular size markers were available in the Source data file.
7
Western Blot Analysis of Synaptic Proteins
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JID: NEUPSY [m6+; November 24, 2018; 20:52 ] Tank) on 10% acrylamide gels and transferred onto nitrocellulose membranes (BIORAD Turbo). Membranes were blocked in Tris-buffered saline, containing 5% nonfat dried milk powder and 0.2% Tween-20 before incubation with the following antibodies: Tau5 (1:2000; Abcam, Cambridge, UK), PHF1 (p-Ser396/404-Tau; 1:200; kindly provided Dr.
Peter Davies (Albert Einstein College of Medicine, New York, NY), GluNR2B (1:1000; Abcam), GluA2 (1:500; Abcam), PSD95 (1:5000; NeuroMab, Davis, CA, USA), Synaptophysin (1:5000; Synaptic Systems, Goettingen, Germany), Synapsin I (1:5000; Synaptic Systems), and 5-HT 1A receptor (1:2500), as well as GAPDH (1:1000; Santa Cruz Biotechnology, Heidelberg, Germany). The Western blots were scanned and quantified using TINA 3.0 bioimaging software (Raytest, Straubenhardt, Germany) after ascertaining linearity. All values obtained for proteins of interest were normalized to levels of GAPDH and expressed as percentages of control male animals; however, pTau levels were normalized to total Tau levels.
Peter Davies (Albert Einstein College of Medicine, New York, NY), GluNR2B (1:1000; Abcam), GluA2 (1:500; Abcam), PSD95 (1:5000; NeuroMab, Davis, CA, USA), Synaptophysin (1:5000; Synaptic Systems, Goettingen, Germany), Synapsin I (1:5000; Synaptic Systems), and 5-HT 1A receptor (1:2500), as well as GAPDH (1:1000; Santa Cruz Biotechnology, Heidelberg, Germany). The Western blots were scanned and quantified using TINA 3.0 bioimaging software (Raytest, Straubenhardt, Germany) after ascertaining linearity. All values obtained for proteins of interest were normalized to levels of GAPDH and expressed as percentages of control male animals; however, pTau levels were normalized to total Tau levels.
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