Journal: Molecular Neurodegeneration

Article Title: Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-020-00404-5

Figure Lengend Snippet: Novel tau mAbs selectively bind AD-tau compared to tau monomer. a Sandwich ELISA assay comprised of total tau capture antibody K9JA and three distinct tau antigens, AD-tau, AD-P1 PFFs, and tau monomer, detected by novel tau mAbs DMR7 or SKT82 and total tau antibody Tau5 as a loading control demonstrating similar levels of captured antigen for each form of tau. b Dot blot assay of AD-tau and tau monomer immobilized onto nitrocellulose membrane without treatment or denatured by guanidine hydrochloride and heat treatment. Total tau immunoblotted by K9JA shows similar levels of tau immobilization. DMR7 and SKT82 selectively detect AD-tau compared to monomer and binding is diminished by denaturation, demonstrating that the conformation of pathological AD-tau is responsible for enhanced binding

Article Snippet: Rabbit polyclonal anti-tau antibody K9JA (Dako) was coated onto 384-well MaxiSorp plates (Fisher Scientific) at 100 ng/well in 0.1 M sodium carbonate buffered to pH 9.6 at 4 °C overnight.

Techniques: Sandwich ELISA, Dot Blot, Binding Assay

Journal: Molecular Neurodegeneration

Article Title: Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-020-00404-5

Figure Lengend Snippet: DMR7 and SKT82 bind to discontinuous epitopes of tau. a Western blot of tau fragments with DMR7 and SKT82 reveal distinct partial binding patterns to tau fragments. DMR7 and SKT82 detect full length tau isoforms T44, T43, and T44, but not the microtubule binding domain, K18. Loss of the C-terminus in the ABP construct and loss of the proline rich domain in the ΔK18-P construct reduce binding of DMR7 and SKT82, demonstrating a proline-rich domain and c-terminal epitope. Equal loading of tau protein fragments was determined by Coomassie blue stained gel and K9JA total tau antibody, which does not detect the ABP fragment. b Schematic of tau constructs and tau mAb binding

Article Snippet: Rabbit polyclonal anti-tau antibody K9JA (Dako) was coated onto 384-well MaxiSorp plates (Fisher Scientific) at 100 ng/well in 0.1 M sodium carbonate buffered to pH 9.6 at 4 °C overnight.

Techniques: Western Blot, Binding Assay, Construct, Staining

Journal: Molecular Neurodegeneration

Article Title: Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-020-00404-5

Figure Lengend Snippet: Tau mAbs inhibit AD-tau seeded aggregation of endogenous mouse tau in primary neurons. a Quantification of immunocytochemistry detection of AD-tau seeded insoluble mouse tau in primary neurons detected by the mouse-tau specific R2295M antibody. Statistical significance was determined relative to non-specific IgG control, using one-way ANOVA with Dunnett’s post-hoc analysis; * p < 0.05, ** p < 0.01, n = 3–4 biological replicates each consisting of 3 technical replicate wells per plate. b Representative immunofluorescent staining images of insoluble mouse tau aggregates in primary neurons induced by AD-tau seeding detected by the mouse tau specific rabbit polyclonal antibody R2295M (green) and DAPI (blue). Tau5, DMR7, and SKT82 show inhibition of seeded tau pathology. c Confocal microscopy images of AD-tau induced aggregates of mouse tau in primary neurons detected by the mouse tau specific antibody T49 (green) and dendritic processes stained with MAP2 (red) with nuclei stained with DAPI (blue). Areas of MAP2 and mouse tau aggregate colocalization are indicated by white arrows. d Immunoprecipitation of tau from AD-tau extracts by IgG, SKT82, or DMR7. Bound and unbound fractions evaluated by western blot with total tau antibody (17025)

Article Snippet: Rabbit polyclonal anti-tau antibody K9JA (Dako) was coated onto 384-well MaxiSorp plates (Fisher Scientific) at 100 ng/well in 0.1 M sodium carbonate buffered to pH 9.6 at 4 °C overnight.

Techniques: Immunocytochemistry, Staining, Inhibition, Confocal Microscopy, Immunoprecipitation, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-020-00404-5

Figure Lengend Snippet: Tau mAbs inhibit uptake of tau seeds into primary neurons. a Western blot characterization of AD-tau seeded fibrillization reaction sedimentation following centrifugation of unlabeled tau monomer (T40 AD-P1), pHR-T40 AD-P1 fibrillization reaction, and pHR-T40 monomer lacking AD-tau seeds. T40 tau monomer and AD-tau are included as controls. b Immunocytochemistry staining of mouse-tau specific R2295M antibody (green) demonstrating that pHR-T40 AD-P1 PFFs are capable of seeding pathological tau aggregates in WT primary mouse neurons. c Sandwich ELISA assay of pHR-T40 AD-P1 and T40 AD-P1 PFFs captured by total tau antibody K9JA and detected with varying concentrations of SKT82 or DMR7. d Immunofluorescence of internalized tau fibrils labeled with pH-sensitive pHRodo-red dye that fluoresces in acidic late endo/lysosomal compartments. Left panels: overlay of brightfield, pHR-T40 AD-P1 PFFs red channel, and DAPI nuclei blue channel. Right panelsl: pHR-T40 AD-P1 PFFs red channel converted to white for visualization. e Quantification of fluorescent internalized pHRodo-red-labeled tau fibrils. Non-specific mouse IgG control did not inhibit uptake of fibrils into neurons, whereas SKT82 and DMR7 significantly inhibited the uptake of fibrils into neurons. One-way ANOVA with Dunnett’s post-hoc analysis **p < 0.01 compared to IgG control n = 4 biological replicates

Article Snippet: Rabbit polyclonal anti-tau antibody K9JA (Dako) was coated onto 384-well MaxiSorp plates (Fisher Scientific) at 100 ng/well in 0.1 M sodium carbonate buffered to pH 9.6 at 4 °C overnight.

Techniques: Western Blot, Sedimentation, Centrifugation, Immunocytochemistry, Staining, Sandwich ELISA, Immunofluorescence, Labeling

Journal: Molecular Neurodegeneration

Article Title: The release and trans-synaptic transmission of Tau via exosomes

doi: 10.1186/s13024-016-0143-y

Figure Lengend Snippet: Tau aggregates are preferentially released via exosomes by a cell model of tauopathy, which can induce aggregation of Tau in N2a cells. Tet-inducible N2a cells were induced to express Tau RDΔK for 2 days by addition of doxycyclin. Then the cells were harvested for sarkosyl extraction to separate soluble Tau and insoluble Tau. The conditioned medium was collected for isolation of exosomes. S and P denote supernatant and pellet of sarkosyl extraction respectively. a Tau RDΔK aggregates in N2a cells and in exosomes. Soluble Tau and Tau aggregates in N2a cells expressing Tau RDΔK or exosomes from this cell model were separated by sarkosyl extraction. The protein loading ratio between supernatant and pellet for N2a cells was 1:60, for exosomes was 1:9. Note that fragment F2 and F3 were detected in pellet of cell extracts but not in pellet of exosomal extracts. b Quantification of Tau RDΔK shown in ( a ). Note that much more Tau RDΔK aggregates were detected in exosomes than in N2a cells. Error bars: SD; n = 3. Student t-test: ** p < 0.01. c Analysis of isolated exosomes separated by sucrose gradient centrifugation. Tau RDΔK appears in fraction 4, coincident with the exosomal marker Alix. d Analysis of sarkosyl pellet of cell lysates separated by sucrose gradient centrifugation. Tau RDΔK is enriched in fraction 1 and 9, but not in fraction 4-- the fraction shown to contain exosomes in ( c ). This indicates that the presence of Tau RDΔK in exosomes is not due to the contamination of Tau aggregates. e Nanotracking analysis of isolated exosomes. The size distribution peaks at ~100 nm, which is typical for exosomes. f Atomic force microscopy of isolated exosomes. The diameters of the majority of the vesicles are 40–100 nm. The average diameter of the exosomes isolated from N2a cells expressing Tau RDΔK is 67.2 ± 15.3 nm and the average height is 2.2 ± 0.8 nm. The exosomes appear to be disk-like shaped. g Cryo-electron tomography of isolated exosomes (compare Fig. ). h Induction of aggregation of full-length Tau ΔK in N2a cells by exosomes from N2a cells expressing Tau RDΔK . N2a cells were transfected with pro-aggregant Tau ΔK and induced to express the protein for 1 day. Then the cells were treated with exosomes (20 μg) derived from N2a cells expressing Tau RDΔK and continuously induced to express Tau ΔK for additional 2 days. Tau ΔK was labeled with K9JA antibody ( red ). Thioflavine S staining was performed to monitor Tau aggregates ( green, arrow ). Hoechst staining was used to label nuclei ( blue ). Note that ThS positive cells were detected in N2a cells (~20-30/3-4X10 4 cells, repeated 3 times) treated with exosomes containing Tau RDΔK , but not in N2a cells treated with broken exosomes. Arrow indicates Tau aggregates. Scale bars = 10 μm

Article Snippet: Rabbit polyclonal antibody K9JA was purchased from Dako (Dako, Glostrup, Denmark).

Techniques: Isolation, Expressing, Gradient Centrifugation, Marker, Microscopy, Tomography, Transfection, Derivative Assay, Labeling, Staining

Journal: Molecular Neurodegeneration

Article Title: The release and trans-synaptic transmission of Tau via exosomes

doi: 10.1186/s13024-016-0143-y

Figure Lengend Snippet: Tau is localized inside exosomes. a Neuron-derived exosomes were incubated with increasing concentrations of NaCl to detach proteins peripherally attached to the membrane. Tau is detected with the pan-Tau antibody K9JA. HSC70 and Alix were examined as exosomal markers. Lines on the right indicate Tau protein, Alix and HSC70. M.W. markers are shown on the left. Note that exosomal Tau levels are not changed by NaCl treatment (lanes 1–5), similar to exosomal markers HSC70 and Alix, indicating that Tau is not peripherally attached to the exosomal membrane surface. b Proteinase K protection assay. Neuron-derived exosomes treated with or without 50 ng proteinase K (Prot K) in the presence or absence of 1% saponin (Sapo) for 5 min or 1 h at 37 °C, followed by western blot analysis. Note that Tau is strongly reduced (5 min) or even absent (1 h) in exosomes treated with both Prot K and Sapo, compared with treatment with Prot K alone, indicating that the exosomal membrane protects Tau against Prot K digestion

Article Snippet: Rabbit polyclonal antibody K9JA was purchased from Dako (Dako, Glostrup, Denmark).

Techniques: Derivative Assay, Incubation, Western Blot

Journal:

Article Title: Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

doi: 10.1091/mbc.02-03-0046

Figure Lengend Snippet: Neurite outgrowth by N2a cells and dependence on tau phosphorylation. N2a cells were differentiated by serum withdrawal and retinoic acid and then transiently transfected with tau or tau mutants, and scored for the development of extended neurites longer than two cell body diameters (>40 μm). (a and c) Tau staining (antibody K9JA). (b and d) Tubulin staining (DM1A). (a and b) Cells transfected transiently with htau23 (50% efficiency). Among the cells not expressing exogenous tau, only 20% develop extended neurites (control; Figure ​Figure2e).2e). In contrast, among the cells expressing exogenous htau23 the fraction with extended neurites rises to 60% (Figure ​(Figure2,2, a and b, arrows, and f). (c and d) Cells transfected with the KXGA mutant of htau23: Among the cells expressing exogenous tau mutant (three cells highlighted by arrows in 2, c and d) only few develop neurites longer than two cell body diameters (compare other untransfected cells in d). This shows that the phosphorylation at KXGS motifs is important for neurite outgrowth. (e) Differentiated control N2a cells (no transfection with tau). (f) Histogram of neurite formation with different tau constructs: Only 20% of control cells show extended neurites, but 60% of cells transfected with htau23. Cells transfected with the KXGA mutant of htau23 are comparable with controls (20%, no phosphorylation by MARK2 possible). Cells transfected with the AP mutant are comparable with transfection by htau23 (60%, no phosphorylation by proline-directed kinases). The data show that transfection with htau23 and the AP mutant are similarly efficient in promoting neurite outgrowth, whereas transfection with the KXGA mutant has no effect. Error bars show SE.

Article Snippet: Immunofluorescence Cells were washed in MTSB buffer (80 mM HEPES, pH 6.9, 1 mM MgCl 2 , 1 mM EGTA, 4% polyethylene glycol) and subsequently fixed with methanol at −20°C for 5 min, washed with phosphate-buffered saline, and treated with 5% bovine serum albumin in phosphate-buffered saline and 0.1% Triton X-100 for 1 h. Fixed cells were incubated with rabbit polyclonal pan-tau antibody K9JA (1:500; Dako Diagnostika), rat monoclonal anti-tubulin antibody YL1/2 (1:200; Serotec, Oxford, United Kingdom), mouse monoclonal anti-hemagglutinin (HA) tag antibody 12CA5 (1:200; Roche Applied Science) or rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR).

Techniques: Transfection, Staining, Expressing, Mutagenesis, Construct

Journal:

Article Title: Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

doi: 10.1091/mbc.02-03-0046

Figure Lengend Snippet: Inhibition of neurite outgrowth by inactive MARK2 (dnMARK2) or nonphosphorylatable Tau (KXGA). N2a cells stably transfected with htau40 can be differentiated by serum withdrawal and retinoic acid (see b and c, asterisks). However, dnMARK2 prevents neurite formation (see a, arrows). (d) Histogram showing that dnMARK2 suppresses neurites (ca. 90% of transfected cells fail to develop neurites). (a–c) Expression of a dominant negative mutant (dnMARK2) in N2a/htau40 cells. (a) Staining for dnMARK2 (antibody 12CA5). (b) Staining for tau (K9JA). (c) Staining for tubulin (DM1A). (e–g) N2a cells cotransfected transiently with GFP-MARK2 and KXGA/htau23 and then differentiated. The doubly transfected cells (arrows) show no neurites, whereas the untransfected cell has extended neurites. (h) Histogram showing the fraction of N2a with extended neurites after transfection: control N2a cells, cells transfected with htau23, MARK2, htau23-KXGA mutant, double transfection with htau23-KXGA and MARK2. The data illustrate that htau23 and MARK2 promote neurite outgrowth, htau23-KXGA abolishes this activation, and even MARK2 is not able to rescue the inhibitory effect of the KXGA-tau mutant on neurite formation after differentiation, because the phosphorylation of tau at its KXGS motifs is blocked.

Article Snippet: Immunofluorescence Cells were washed in MTSB buffer (80 mM HEPES, pH 6.9, 1 mM MgCl 2 , 1 mM EGTA, 4% polyethylene glycol) and subsequently fixed with methanol at −20°C for 5 min, washed with phosphate-buffered saline, and treated with 5% bovine serum albumin in phosphate-buffered saline and 0.1% Triton X-100 for 1 h. Fixed cells were incubated with rabbit polyclonal pan-tau antibody K9JA (1:500; Dako Diagnostika), rat monoclonal anti-tubulin antibody YL1/2 (1:200; Serotec, Oxford, United Kingdom), mouse monoclonal anti-hemagglutinin (HA) tag antibody 12CA5 (1:200; Roche Applied Science) or rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR).

Techniques: Inhibition, Stable Transfection, Transfection, Expressing, Dominant Negative Mutation, Staining, Mutagenesis, Activation Assay

Journal:

Article Title: Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

doi: 10.1091/mbc.02-03-0046

Figure Lengend Snippet: Phosphorylation of tau induced by MARK2. (a–c) N2a/htau40 cells transiently transfected with GFP-MARK2, differentiated, fixed after 24 h, and analyzed by fluorescence microscopy. (a) Distribution of GFP-MARK2. (b) Immunofluorescence of p-MARK antibody against active MARK2 (phosphorylated T-loop). (c) Antibody 12E8 against phospho-KXGS motifs of tau. Note that active MARK2 and phosphorylated tau at KXGS sites colocalize (see below). (d and e) N2a/htau40 cells were transiently transfected with MARK2 or a dominant negative mutant (dnMARK2), differentiated for 6 h, and analyzed by preparing a cell lysate and probing by Western blotting with a panel of antibodies against phosphorylated MARK2 and tau. The data show that MARK2 causes the phosphorylation of the KXGS motifs of tau and dnMARK2 inhibits this. (d) Lane 1, control N2a/htau40 cell extract. Lane 2, transfection with MARK2. Lane 3, transfection with dominant negative MARK2 (T208A and S212A). Antibody K9JA (“Tau”) stains tau independently of phosphorylation. The three samples contain the same amount of tau. Antibody 12E8 against the phosphorylated KXGS motifs in tau repeats 1 and 4 (phospho-S262 and -S356, “p-Tau”) shows stronger staining in the case of MARK2 transfection (lane 2), but only weak staining without MARK2 transfection (lane 1), and no staining with dnMARK2 (lane 3). The antibody against hemagglutinin-tag (“MARK”) reveals the expression of exogenous MARK2 or dnMARK2 (lanes 2 and 3) but is absent from untransfected cells (lane 1). The antibody SA6941 against phosphorylated T-loop peptide on MARK2 (pT208, pS212, corresponding to activated MARK2) shows pronounced staining after transfection of exogenous MARK2 (lane 2) but only weak staining in controls (lane 1), corresponding to the activity of endogenous MARK-like kinases, and no staining in the presence of dnMARK. (e) N2a/htau40 cells were transiently transfected with MARK2 or a dominant negative mutant, dnMARK2, differentiated for 6 h, and before harvesting treated for 30 min with 0.2 μM okadaic acid. Note that only the sample expressing the transfected wt MARK2, but not dnMARK2, has clearly increased phosphorylation at KXGS motifs shown by reaction with antibody 12E8 (“p-Tau”, lane 2). This means that MARK2 but not other endogenous kinases such as PKA are responsible for tau phosphorylation at KXGS motifs in these conditions. (f and g) Western blot analysis demonstrating the specificity of the p-MARK antibody (polyclonal antibody SA6941) against the posphorylation sites T208 and S212 in the activating loop of kinase MARK2. Top, MARK2 protein before (lane 1) and after (lane 2) phosphorylation by brain extract kinase activity (see MATERIALS AND METHODS). Bottom, antibody p-MARK (SA6941) recognizes exclusively the phosphorylated MARK2 (lane 2) whose increased activity is demonstrated in the histogram (g). (h–j) N2a/htau40 cells transiently transfected with HA-tagged MARK2, fixed 24 h after differentiation and analyzed by confocal fluorescence microscopy. (h) Distribution of MARK2 by using fluorescent HA antibody. (i) Fluorescence of phalloidin-labeled actin. (j) Merge of h and i. Note that MARK2 and actin largely colocalize.

Article Snippet: Immunofluorescence Cells were washed in MTSB buffer (80 mM HEPES, pH 6.9, 1 mM MgCl 2 , 1 mM EGTA, 4% polyethylene glycol) and subsequently fixed with methanol at −20°C for 5 min, washed with phosphate-buffered saline, and treated with 5% bovine serum albumin in phosphate-buffered saline and 0.1% Triton X-100 for 1 h. Fixed cells were incubated with rabbit polyclonal pan-tau antibody K9JA (1:500; Dako Diagnostika), rat monoclonal anti-tubulin antibody YL1/2 (1:200; Serotec, Oxford, United Kingdom), mouse monoclonal anti-hemagglutinin (HA) tag antibody 12CA5 (1:200; Roche Applied Science) or rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR).

Techniques: Transfection, Fluorescence, Microscopy, Immunofluorescence, Dominant Negative Mutation, Western Blot, Staining, Expressing, Activity Assay, Labeling

Journal:

Article Title: Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

doi: 10.1091/mbc.02-03-0046

Figure Lengend Snippet: Effect of kinase inhibitors FL and HD on tau-induced neurite outgrowth in N2a cells stably transfected with htau40. (a) Control N2a/htau40 cells were differentiated and stained for immunofluorescence with tau antibody K9JA; ∼25% of cells develop extended cell processes. (b) N2a/htau40 cells differentiated and treated with 50 μM FL, a strong inhibitor of cdk5 and GSK-3β (Leclerc et al., 2001 ). Neurite outgrowth is somewhat enhanced (∼35%). (c) N2a/htau40 cells differentiated and treated with 50 μM HD, also a strong inhibitor of cdk5 and GSK-3β (Meijer et al., 2000 ). Neurite outgrowth is strongly inhibited (∼3%). (d) Histogram illustrating the fraction of N2a/htau40 cells bearing extended neurites after differentiation and treatment with kinase inhibitors HD and FL. HD strongly reduces the extent of neurite formation, and FL causes a slight increase.

Article Snippet: Immunofluorescence Cells were washed in MTSB buffer (80 mM HEPES, pH 6.9, 1 mM MgCl 2 , 1 mM EGTA, 4% polyethylene glycol) and subsequently fixed with methanol at −20°C for 5 min, washed with phosphate-buffered saline, and treated with 5% bovine serum albumin in phosphate-buffered saline and 0.1% Triton X-100 for 1 h. Fixed cells were incubated with rabbit polyclonal pan-tau antibody K9JA (1:500; Dako Diagnostika), rat monoclonal anti-tubulin antibody YL1/2 (1:200; Serotec, Oxford, United Kingdom), mouse monoclonal anti-hemagglutinin (HA) tag antibody 12CA5 (1:200; Roche Applied Science) or rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR).

Techniques: Stable Transfection, Transfection, Staining, Immunofluorescence

Journal:

Article Title: Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

doi: 10.1091/mbc.02-03-0046

Figure Lengend Snippet: Effect of kinase inhibitors on phosphorylation of htau23 in Sf9 cells. (a and b) Antibody blots. (c and f) 2D phosphopeptide maps. (a) Lane 1, no inhibitor (control). Lane 2, 50 μM HD. Lane 3, 50 μM FL. From top to bottom: Coomassie-stained gel (control) showing roughly equal amounts of tau; Western blot with antibody K9JA that recognizes tau independently of phosphorylation; antibody 12E8 (against phosphorylated KXGS motifs). The inhibitor HD prevents phosphorylation (lane 2) because it inhibits MARK and related kinases, and not because of its inhibition of GSK-3β (see below). LiCl and PKA inhibitors have no pronounced effect (Figure ​(Figure8b,8b, lanes 1 and 2). Antibody AT-100 (against phospho-T212 and S214). This epitope requires the activity of GSK-3β (to phosphorylate T212), and PKA (to phosphorylate S214; Zheng-Fischhofer et al., 1998 ). Therefore, the epitope is blocked by both HD and FL (lanes 2 and 3). Antibody AT-180 (against phospho-T231 and S235). This is an epitope induced by cdk5 and GSK-3β, and thus the signal is blocked by both HD and FL (lanes 2 and 3). Antibody AT-8 (against phospho-S202 and T205). This is an epitope induced mainly by cdk5, and therefore the signal is blocked by both HD and FL (lanes 2 and 3). Antibody PHF-1 (against phospho-S396 and S404). This epitope is phophorylated mainly by GSK-3β, and therefore the signal is inhibited by HD, but less by FL. (b) Lane 1, 50 mM LiCl. Lane 2, 50 μM H89, a PKA inhibitor. (c–f) Two-dimensional phosphopeptide maps of htau23 protein expressed in Sf9 cells and phosphorylated by endogenous kinases without (c), or in the presence of kinase inhibitors (d–f). (c) Control, tau expressed in Sf9 cells. (d) Inhibition by 50 μM HD to inhibit cdk5, GSK-3β, and MARK. (e) Inhibition by 50 mM LiCl to inhibit GSK-3β. (f) Inhibition by 50 μM FL to inhibit cdk5 and GSK3β; note that c, e, and f show spots for phosphorylated S262 (circle, also see insert in e at higher exposure), d does not because HD inhibits MARK activity.

Article Snippet: Immunofluorescence Cells were washed in MTSB buffer (80 mM HEPES, pH 6.9, 1 mM MgCl 2 , 1 mM EGTA, 4% polyethylene glycol) and subsequently fixed with methanol at −20°C for 5 min, washed with phosphate-buffered saline, and treated with 5% bovine serum albumin in phosphate-buffered saline and 0.1% Triton X-100 for 1 h. Fixed cells were incubated with rabbit polyclonal pan-tau antibody K9JA (1:500; Dako Diagnostika), rat monoclonal anti-tubulin antibody YL1/2 (1:200; Serotec, Oxford, United Kingdom), mouse monoclonal anti-hemagglutinin (HA) tag antibody 12CA5 (1:200; Roche Applied Science) or rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR).

Techniques: Staining, Western Blot, Inhibition, Activity Assay