total pp1  (Santa Cruz Biotechnology)

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1 is upstream of eIF2-α phosphorylation in the cell death induced by the combination of OSU-03012 and lapatinib. A : BT474 cells (5 x 10 5 ) were co-transfected with the Ser 51 Ala eIF2-α mutant and GFP-Nck1. Cells were allowed to incubate for 24 h to induce ectopic expression then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested for western blotting as described previously (lower panel), or plated into 6-well plates to assay for clonogenic capacity as described previously (upper graph). Graphs represent total colony number. * denotes a p-value of <0.05. B : BT474 (upper panel) and MDA-MB-231 (lower panel) cells were treated with either vehicle (Veh) or OSU-03012 and lapatinib in combination (combo). PP1 was immunoprecipitated and the resulting membranes were immunoblotted with eIF2α and PP1. C : Our data indicate that Nck1 and PP1, which are originally in a complex with eIF2 are dissociated after treatment with the combination of OSU-03012 and lapatinib. This event frees eIF2-α to become phosphorylated by one of many upstream kinases such as PERK, leading to ER stress and eventual cell death.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Transfection, Mutagenesis, Expressing, Western Blot, Immunoprecipitation

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Phosphorylation of eIF2-α indicates ER stress signaling in MDA-MB-231 and BT474 cells after treatment with OSU-03012 and lapatinib. MDA-MB-231 cells and BT474 cells (1 x 10 6 ) were subjected to vehicle (DMSO, Ctr), OSU-03012 (2 μM), lapatinib (2 μM) or the combination as indicated for 3 h. Cells were then lysed and protein extracts (10-20 μg) were subjected to SDS PAGE followed by western immunoblotting for the indicated proteins.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: SDS Page, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: The role of eIF2-α phosphorylation in cell death induced by OSU-03012 and lapatinib. A-B : MDA-MB-231 ( A ) or BT474 ( B ) cells (5 x 10 5 ) were transfected with the indicated siRNA molecules and incubated for 48 h. Cells were then treated with either vehicle (Veh) or the combination of OSU-03012 (2 μM) and lapatinib (2 μM) (combo) as indicated and either subjected immunoassays (bottom panels) or plated for clonogenic capacity (top graphs). Numbers indicated are percent control (e.g. Veh). C-D : MDA-MB-231 cells (5 x 10 5 ) were transfected with a control vector (Vector), wild-type (WT) or the Ser 51 Ala mutant (S51A) eIF2-α plasmids. After a further 24 h cells were plated onto 6-well plates to assay for clonogenic capacity ( D ) or subjected to immunoblotting as described in Materials and Methods ( C ). Cells were treated with either vehicle (Veh, DMSO) or OSU-03012 (2 μM) and lapatinib (2 μM) in combination (combo) for 24 h ( D ) or 3 h ( C ). Colonies were allowed to develop for the next 10-14 days. * indicates a p < 0.05 with respect to vehicle-treated cells. E : MDA-MB-231 cells (1 x 10 6 ) were plated and treated with the indicated drugs (Vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo)). Three hours later, cells were harvested and subjected to immunoblotting. Samples were probed with the indicated antibodies (see Materials and Methods).

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Transfection, Incubation, Plasmid Preparation, Mutagenesis, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Upstream signaling responsible for eIF2-α phosphorylation: A role for Nck1. A-B : MDA-MB-231 cells and BT474 cells were treated with vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo) for 3 h, then harvested for immunoblotting assays as described in Materials and Methods. Membranes were probed with the indicated antibodies. C : MDA-MB-231 cells (5 x 10 5 ) were transfected with plasmids to express either GFP-Nck1 or GFP-Nck2 as described. After an additional 24 h, cells were treated with the combination of OSU-03012 and lapatinib as indicated for 24 h (upper graphs) or 3 h (lower panels), and then either plated onto 6-well dishes and allowed to form colonies (graphs represent percent control) or harvested for immunoblotting assays and probed with the indicated antibodies. * indicates a p<0.05.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Western Blot, Transfection

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1, but not Nck2 expression ablates the increase in cell death induced by OSU-03012 and lapatinib. A-D : MDA-MB-231 cells ( A, B ), or BT474 cells ( C, D ) (5 x 10 5 ) were co-transfected with either wild-type eIF2-α and the two Nck isoforms ( A, C ), or the S 51 A phospho-mutant and the two Nck isoforms ( B, D ). Cells were allowed to incubate for 24 h to induce ectopic expression, and then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested after a 3 h treatment for western blotting as described in Materials and Methods (bottom panels), or plated singly onto 6-well plates to assay for clonogenic capacity (upper graphs). Cells were treated with OSU-03012 (2 μM) and lapatinib (2 μM) for 24 h, and then allowed to form colonies for 10-14 days. Total colony counts were graphed. * denotes a p-value of <0.05.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Expressing, Transfection, Mutagenesis, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1 is upstream of eIF2-α phosphorylation in the cell death induced by the combination of OSU-03012 and lapatinib. A : BT474 cells (5 x 10 5 ) were co-transfected with the Ser 51 Ala eIF2-α mutant and GFP-Nck1. Cells were allowed to incubate for 24 h to induce ectopic expression then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested for western blotting as described previously (lower panel), or plated into 6-well plates to assay for clonogenic capacity as described previously (upper graph). Graphs represent total colony number. * denotes a p-value of <0.05. B : BT474 (upper panel) and MDA-MB-231 (lower panel) cells were treated with either vehicle (Veh) or OSU-03012 and lapatinib in combination (combo). PP1 was immunoprecipitated and the resulting membranes were immunoblotted with eIF2α and PP1. C : Our data indicate that Nck1 and PP1, which are originally in a complex with eIF2 are dissociated after treatment with the combination of OSU-03012 and lapatinib. This event frees eIF2-α to become phosphorylated by one of many upstream kinases such as PERK, leading to ER stress and eventual cell death.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Transfection, Mutagenesis, Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: P301L tau increases the association with PP1 in primary hippocampal neurons. A , Lentiviruses were used to express WT, P301L, or R5L tau in cultured primary rat hippocampal neurons beginning at DIV 4 and fixed in 4% paraformaldehyde on DIV 8. The neurons were probed with Tau7 (C-terminal tau epitope) and a PP1 antibody before performing the PLA staining procedure that demonstrates a close association between these proteins (<40 nm) indicated by a green fluorescent punctate signal. Following PLA, the cells underwent immunocytofluorescence staining with a Tau12 (red, human tau-specific N-terminal tau epitope) to identify exogenous tau and Tuj1 (cyan) to identify neurons and neuron processes. B , The PP1 antibody recognizes recombinant PP1α, PP1β, and PP1γ in an immunoblot (green), and a 6× His antibody labels total protein. C , Images of isolated neurons were used to quantify puncta per area of Tau12 staining (i.e., transduced neurons). P301L tau-expressing neurons (67.3 ± 17.7 puncta/nm 2 ) showed significantly greater PLA signal than WT (23.1 ± 8.5 puncta/nm 2 ) and R5L tau-expressing neurons (31.3 ± 9.8 puncta/nm 2 ), complementing results obtained in HEK cell pull down and NanoBRET assays ( , ). Data represent mean ± SD and were analyzed using repeated measures one-way ANOVAs and Tukey's post hoc multiple comparison test, * p ≤ 0.05. D–F , PLA detected associations between WT tau and endogenous rat PP1 (PLA, green puncta; Tau12, red), in both neuronal processes ( E ) and cell bodies ( F ). G–I , Neurons expressing P301L tau displayed an enhanced association between tau and PP1 as evidenced by the PLA puncta, which were found in processes ( H ) and cell bodies of neurons ( I ). J–L , Neurons expressing R5L tau showed an association between tau and PP1 that was similar to WT tau cells and localized to neuronal processes ( K ) and cell bodies ( L ). M–O , Primary antibody delete controls included omission of either Tau7 ( M ), PP1 ( N ), or both ( O ). Primary delete controls showed little to no nonspecific background signal. P–R , PLA in untransduced neurons (No LV) demonstrated an association between endogenous rat tau and PP1, which were evident in neuronal processes ( Q ) and cell bodies ( R ) of neurons. Scale bars: 20 μm for all images.

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Cell Culture, Staining, Recombinant, Western Blot, Isolation, Expressing

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: PP1γ knockdown rescues the effects of P301L and R5L on fast axonal transport pause frequency in primary neurons. PP1 expression was knocked down using isoform-specific shRNAs. The shRNAs were validated using a Renilla -Firefly Luciferase assay in HEK293T cells. Renilla -PP1 fusion constructs were cotransfected with shRNAs targeting Renilla (positive control), a nontargeting shRNA sequence (Control shRNA; used as a negative control), or specific PP1 isoforms. Expression knockdown is reported as percentage of an empty shRNA vector control. A , PP1α shRNA produced 87% knockdown from control levels. B , PP1γ shRNA induced 90% knockdown from control levels. Data were compared ( A–B ) using a one-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent replicate ( n = 3; data are mean ± SD; * p ≤ 0.05). C , Neuronal mRNA knockdown was validated using lentiviral expression of PP1α-shRNA, PP1γ-shRNA, or the control shRNA. PP1α and PP1γ mRNA concentrations were determined using ddPCR and normalized to rplp0 reference gene expression. PP1α shRNA treatment knocked down PP1α mRNA compared with control shRNA treatment, whereas PP1γ shRNA treatment only minimally reduced it. D , PP1γ shRNA knocked down PP1γ mRNA compared with control shRNA treatment, whereas PP1α shRNA slightly reduced it. Error bars in C and D represent the 95% confidence intervals. E , Transport experiments were repeated with PP1α, PP1γ-, or control shRNA. Anterograde segment velocity was not significantly different among any of the conditions. Dotted lines represent the baseline values (i.e., the mean of values in the WT tau + control shRNA treatment condition for E and F . F , Retrograde segment velocity was slightly higher with R5L expression compared with WT tau with control shRNA. G , Expressing P301L tau or R5L tau increased total pause frequency compared with WT tau when treated with control or PP1α-shRNA. In contrast, PP1γ-shRNA treatment rescued P301L- and R5L-induced increases in pause frequency by reducing total pause frequency to WT tau control levels. H , P301L and R5L tau increased anterograde pause frequency compared with WT tau + control or PP1α-shRNA. Treatment with PP1γ-shRNA reduced anterograde pause frequency in P301L and R5L neurons compared with WT tau neurons. I , P301L and R5L expression increased retrograde pause frequency compared with WT tau with control shRNA treatments. Coexpression of WT, P301L, or R5L tau with PP1α-shRNA were not different likely because of the enhanced pause frequency in WT. With P301L and R5L, PP1γ-shRNA reduced retrograde pause frequency to levels similar to WT with PP1γ-shRNA and to baseline levels (i.e., WT with control shRNA). Data were compared ( E–I ) using a two-way ANOVA with Tukey's multiple comparison test to compare the three tau groups within each individual shRNA treatment group, and each data point represents an independent replicate ( n = 6; the data are mean ± SD; * p ≤ 0.05; , mean ± SD; , for statistical p values).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Expressing, Luciferase, Construct, Positive Control, shRNA, Sequencing, Negative Control, Plasmid Preparation, Produced

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: FTLD mutant tau enhances the levels of active PP1 in cells and primary neurons. A , Immunoblotting confirmed expression of GFP, WT, P301L, and R5L tau in HEK293T cells coexpressing PP1α. B , Quantification of tau bands indicated similar levels of tau across the three constructs (WT tau, 4.87 ± 0.36; P301L, 5.73 ± 0.58; R5L: 5.67 ± 0.66). C , Immunoblots of total PP1 (green, HaloTag antibody) and inactive PP1 (red, phospho-Thr320 PP1 antibody). D , Quantification of PP1 blots showed that P301L tau increased levels of active PP1α (as indicated by reduced inactive pT320 PP1 signal) compared with GFP control (GFP, 0.731 ± 0.331; WT tau, 0.627 ± 0.054; P301L, 0.388 ± 0.066; R5L, 0.409 ± 0.121). E , Immunoblots of GFP and tau in cell lysates coexpressing PP1β. F , Quantification of tau bands indicated similar expression of tau (WT tau, 7.58 ± 1.66; P301L, 7.94 ± 1.15; R5L, 9.72 ± 3.72). G , Immunoblots of total PP1 (green) and inactive pT316 PP1β (red). H , Quantification of PP1 blots showed that expression of any of the three forms of tau significantly increased active PP1β levels (i.e., reduced inactive pT316 PP1β) compared with GFP control (GFP, 0.0155 ± 0.0017; WT tau, 0.0123 ± 0.0019; P301L, 0.0084 ± 0.0013; R5L, 0.0065 ± 0.0003). P301L and R5L tau significantly increased active PP1β compared with WT tau as well. I , Immunoblots of cells coexpressing GFP, WT tau, P301L tau, or R5L tau and PP1γ. J , Quantification of tau bands showed similar tau expression levels (WT tau, 13.4 ± 3.3; P301L, 15.9 ± 3.4; R5L, 21.6 ± 11.8). K , Immunoblots of total PP1 (green) and inactive pT311 PP1γ (red). L , Quantification of PP1 blots demonstrated that expression of all forms of tau increased active PP1γ (i.e., reduced inactive pT311 PP1γ; GFP, 0.0453 ± 0.0142; WT tau, 0.0161 ± 0.0030; P301L, 0.0045 ± 0.0013; R5L, 0.0065 ± 0.0026). M , Immunoblot of lysates from rat primary neurons expressing GFP or tau (WT, P301L, or R5L) via lentiviral transduction probed with tau (green) and GFP (red) and a loading control (GAPDH). N , Quantification of the exogenous tau bands (top, green) indicated similar levels of tau across the three constructs (WT tau, 19.2 ± 3.9; P301L, 14.8 ± 2.6; R5L, 17.4 ± 4.6). O , Immunoblot of total PP1 (green, PP1 antibody) and inactive PP1 (red, pThr320 PP1 antibody). P , Quantification of the immunoblots demonstrated that P301L tau and R5L tau expression increased levels of active PP1 (i.e., reduced inactive pT320 PP1) compared with a control GFP expression, whereas WT tau did not significantly change active PP1 levels (GFP, 0.0192 ± 0.0012; WT tau, 0.0190 ± 0.0029; P301L, 0.0159 ± 0.0006; R5L, 0.0162 ± 0.0015). Note that tau signal was normalized to GAPDH loading control ( B , F , J , N ), and pT320 PP1 signal was normalized to total PP1 signal ( D , H , L , P ). Data in figure legend are reported in ratios of the signal intensities in arbitrary units. All data are mean ± SD and were compared using repeated measures one-way ANOVAs with a Geisser–Greenhouse correction and Tukey's post hoc multiple comparison test, * p ≤ 0.05).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Mutagenesis, Western Blot, Expressing, Construct, Transduction

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: Tau-induced increases in active PP1 is dependent on PAD. PAD, a conformationally displayed biologically active motif in the extreme amino terminus of tau, was deleted in WT and P301L tau by removing amino acids 2–18 (Δ2–18). A , B , The full-length and Δ2–18 constructs were expressed in HEK293 cells, in addition to PP1α, at similar levels ( B ; WT tau, 4.02 ± 0.54; Δ2–18 WT, 3.24 ± 0.63; P301L, 5.32 ± 1.52; Δ2–18 P301L, 4.04 ± 0.58). C , Levels of active PP1α (as indicated by changes in inactive pT320 PP1) were identified using a phospho-Thr320 PP1 antibody (red), and total levels were detected using an anti-HaloTag antibody (green). D , Deletion of PAD resulted in significantly lower levels of active PP1 compared with expression of full-length tau in both WT and P301L backgrounds (WT tau, 0.3409 ± 0.0366; Δ2–18 WT, 0.4232 ± 0.0336; P301L, 0.2147 ± 0.0507; Δ2–18 P301L, 0.4062 ± 0.0406). E , F , WT tau, Δ2–18 WT, P301L, and Δ2–18 P301L were expressed at similar levels ( F ) along with PP1β (WT tau, 1.71 ± 0.14; Δ2–18 WT, 1.64 ± 0.28; P301L, 1.68 ± 0.24; Δ2–18 P301L, 1.78 ± 0.41). G , Immunoblotting of total PP1 (green) and inactive PP1β (red, pT320 PP1 antibody corresponds to pT316 in PP1β). H , Active PP1β levels decreased in the presence of Δ2–18 WT and Δ2–18 P301L compared with full-length WT and P301L tau (WT tau, 0.0393 ± 0.0067; Δ2–18 WT, 0.1525 ± 0.0281; P301L, 0.0162 ± 0.0062; Δ2–18 P301L, 0.1231 ± 0.0125). I , WT tau, Δ2–18 WT, P301L, and Δ2–18 P301L in cells coexpressing PP1γ. J , Quantification indicated similar levels of tau expression with all constructs (WT tau, 8.82 ± 1.27; Δ2–18 WT, 7.26 ± 1.57; P301L, 8.32 ± 2.32; Δ2–18 P301L, 6.96 ± 1.73). K , Immunoblots of total PP1 (green) and inactive PP1γ (red, pT320 PP1 antibody corresponds to pT311 in PP1γ). L , The Δ2–18 WT resulted in decreased levels of active PP1γ compared with full-length counterparts (WT tau, 0.0578 ± 0.0099; Δ2–18 WT, 0.1428 ± 0.0154; P301L, 0.0101 ± 0.0022; Δ2–18 P301L, 0.0862 ± 0.0471). P301L tau expression led to higher levels of active PP1 compared with WT tau, and this effect was reduced about ninefold on PAD deletion in Δ2–18 P301L. Tau signal was normalized to GAPDH loading control ( B , F , J ) and pT320 PP1 signal was normalized to total PP1 signal ( D , H , L ). Data in legend are reported in ratios of the signal intensities in arbitrary units. All data in figure are mean ± SD and were compared using repeated measures one-way ANOVAs with a Geisser–Greenhouse correction and Tukey's post hoc multiple comparison test, * p ≤ 0.05).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Construct, Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: P301L tau increases the association with PP1γ isoform in primary hippocampal neurons. A , PLA experiments were repeated with the Tau7 antibody and a PP1γ-specific antibody verified with immunoblot. Recombinant PP1 isoforms were probed with a PP1γ antibody (green) and a 6xHis antibody (red). B , PLA puncta were quantified and normalized to area of Tau12 staining (i.e., exogenous tau staining). P301L tau (28.0 ± 7.0 puncta/nm 2 ) expression increased levels of PLA signal compared with WT tau (11.3 ± 1.9 puncta/nm 2 ), whereas R5L (16.2 ± 2.1 puncta/nm 2 ) expression did not significantly change. Data represent mean ± SD of three replicates and were analyzed using a repeated measures one-way ANOVA and Tukey's post hoc multiple comparison test; * p ≤ 0.05. C , A Tau7 primary antibody deletion demonstrated the lack of background signal when PLA is performed with the PP1γ antibody only. D–F , Associations between WT tau and endogenous rat PP1γ (PLA, green puncta; Tau12, red) were identified in the neuronal processes ( E ) and cell bodies ( F ) of the neurons. G – I , Lentiviral expression of P301L tau increased PLA puncta, which were found in processes ( H ) and cell bodies of neurons ( I ). J–L , PLA in the presence of R5L tau also demonstrated an association between tau and PP1γ, localized to the processes ( K ) and cell bodies ( L ) of the neurons. M–O , PLA was performed in untransduced neurons to identify an association between endogenous rat tau and PP1γ, also found in neuronal processes ( N ) and cell bodies ( O ). Scale bars: 20 μm for all images.

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Western Blot, Recombinant, Staining, Expressing

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: P301L mutation in tau enhances the interaction with PP1α and γ isoforms in pull-down (PD) assays. A , Schematic showing assay design. Individual tau constructs were coexpressed with HaloTag-PP1 (left) or HaloTag-only controls (right) in HEK293T cells. Lysates prepared from these cells were incubated with HaloLink resin, which covalently binds to HaloTag. We quantified the amount of tau signal (pan-tau R1 antibody, green) eluted from each pull down by immunoblot (PD elution) and normalized to the reduction in Halo signal (anti-Halo antibody, red) from prebinding to postbinding lysate samples. B , WT, P301L tau, and R5L tau proteins were detected in the elution samples after a pull down with Halo-PP1α (left, red box). Tau was not detected in the HaloTag-only control (right, red box). Lysate samples obtained before (Input) and after pull down with the HaloLink resin (Post PD) were probed to confirm similar protein expression and HaloTag binding to resin. C , Quantitation of tau in the elution samples indicate that P301L tau (0.673 ± 0.429) significantly increases the interaction with PP1α when compared with WT (0.160 ± 0.147) and R5L tau (0.107 ± 0.041) and the control with HaloTag-only (WT tau, 0.004 ± 0.002; P301L, 0.003 ± 0.003; R5L, 0.003 ± 0.001). D , A relatively low level of WT and P301L tau was eluted from the Halo-PP1β pull down but not the HaloTag-only control. The pre- and post-pull-down lysate samples from the Halo-PP1β and Halo-only lysates confirm similar expression and pull-down efficiency. E , Quantitation of tau in the pull-down elution samples indicate that P301L tau (0.061 ± 0.038) significantly increases the interaction with PP1β when compared with the P301L + HaloTag control (−0.001 ± 0.001). Notably, the amount of tau in the elution with Halo-PP1β pull down is much lower for all forms (WT tau, 0.023 ± 0.024; R5L, 0.009 ± 0.005) and not significantly higher than the HaloTag-only controls (WT tau, 0.001 ± 0.001; R5L, −0.001 ± 0.001). F , WT and P301L tau proteins were detected in the elution samples after a pull down with Halo-PP1γ. The pre- and post-pull-down lysate samples from the Halo-PP1γ and Halo-only lysates confirm similar expression and pull-down efficiency. G , Quantitation of tau in the elution samples indicate that P301L tau (0.540 ± 0.209) significantly increases the interaction with PP1γ when compared with WT tau (0.125 ± 0.069), R5L tau (0.111 ± 0.075), and the P301L + HaloTag control (0.000 ± 0.001). Tau was also absent in HaloTag pull-down controls (WT tau, −0.001 ± 0.002; R5L, 0.001 ± 0.002). All data in the legend are reported as ratios of the signal intensities in arbitrary units. Statistical comparisons were performed using a repeated measures two-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent experimental replicate ( n = 4 independent replicates; data are mean ± SD; * p ≤ 0.05 for comparisons among the tau group; # p ≤ 0.05 for the comparison between Halo-PP1 and Halo-only pull downs).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Mutagenesis, Construct, Incubation, Western Blot, Expressing, Binding Assay, Quantitation Assay

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: Tau protein interacts with PP1α and PP1γ, and the P301L tau mutation enhances the interaction in intracellular nBRET assay. A , Tau proteins were tagged with a luciferase enzyme (NanoLuc) and coexpressed in HEK293T cells in the presence of an individual Halo-PP1 isoform or HaloTag-only control protein. A fluorescent ligand binds to the HaloTag, forming the acceptor molecule. Light emitted from the luciferase (donor) activates the fluorophore (acceptor) if they are within ∼37 Å of each other because of a specific protein–protein interaction. The donor and acceptor signals were measured using filtered luminescence and reported as the nBRET ratio. The acceptor:donor DNA ratio was varied to generate a donor saturation assay curve. B , The hyperbolic shape of the donor saturation curves indicated a specific protein–protein interaction between Halo-PP1α and WT tau (solid black), P301L tau (solid gray), and R5L tau (dashed black) tagged with NanoLuciferase. C , A strong interaction was not detected for the same forms of tau in the presence of Halo-PP1β. D , Each form of tau displayed a clear interaction with Halo-PP1γ. E , There was no evidence of a specific interaction between the Halo-only control and any of the tau proteins. F , The nBRET was performed at an acceptor-to-donor DNA ratio of 100:1 with four independent replicates to compare nBRET ratios among the tau groups with Halo-PP1 or Halo-only. Each tau displayed significantly higher nBRET ratios with Halo-PP1α (WT tau, 9.57 ± 1.05 mBU; P301L, 20.40 ± 2.58 mBU; R5L, 10.25 ± 0.57 mBU) compared with Halo-only controls (WT tau, 5.168 ± 0.16 mBU; P301L, 5.814 ± 0.36 mBU; R5L, 5.418 ± 0.30 mBU). P301L tau nBRET ratios were significantly higher than WT and R5L tau with Halo-PP1α. G , There was no significant increase detected for any of the individual tau constructs with Halo-PP1β (WT tau, 6.067 ± 1.69 mBU; P301L, 8.323 ± 2.59 mBU; R5L, 6.325 ± 1.53 mBU) compared with Halo-only or among the tau constructs and Halo-PP1β. H , The nBRET ratios for each of the tau forms were significantly higher with Halo-PP1γ (WT tau, 9.579 ± 0.429 ± 1.71 mBU; P301L, 20.38 ± 1.99 mBU; R5L, 8.939 ± 1.37 mBU) compared with Halo-only controls. The nBRET ratios between Halo-PP1γ and P301L tau significantly increased compared with WT and R5L tau. Statistical comparisons for F–H were performed using a two-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent experimental replicate (data are mean ± SD; * p ≤ 0.05 for the comparison among the tau groups; # p ≤ 0.05 for the comparison between Halo-PP1 and Halo-only groups).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Mutagenesis, Luciferase, Saturation Assay, Construct

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: P301L tau increases the association with PP1 in primary hippocampal neurons. A , Lentiviruses were used to express WT, P301L, or R5L tau in cultured primary rat hippocampal neurons beginning at DIV 4 and fixed in 4% paraformaldehyde on DIV 8. The neurons were probed with Tau7 (C-terminal tau epitope) and a PP1 antibody before performing the PLA staining procedure that demonstrates a close association between these proteins (<40 nm) indicated by a green fluorescent punctate signal. Following PLA, the cells underwent immunocytofluorescence staining with a Tau12 (red, human tau-specific N-terminal tau epitope) to identify exogenous tau and Tuj1 (cyan) to identify neurons and neuron processes. B , The PP1 antibody recognizes recombinant PP1α, PP1β, and PP1γ in an immunoblot (green), and a 6× His antibody labels total protein. C , Images of isolated neurons were used to quantify puncta per area of Tau12 staining (i.e., transduced neurons). P301L tau-expressing neurons (67.3 ± 17.7 puncta/nm 2 ) showed significantly greater PLA signal than WT (23.1 ± 8.5 puncta/nm 2 ) and R5L tau-expressing neurons (31.3 ± 9.8 puncta/nm 2 ), complementing results obtained in HEK cell pull down and NanoBRET assays ( , ). Data represent mean ± SD and were analyzed using repeated measures one-way ANOVAs and Tukey's post hoc multiple comparison test, * p ≤ 0.05. D–F , PLA detected associations between WT tau and endogenous rat PP1 (PLA, green puncta; Tau12, red), in both neuronal processes ( E ) and cell bodies ( F ). G–I , Neurons expressing P301L tau displayed an enhanced association between tau and PP1 as evidenced by the PLA puncta, which were found in processes ( H ) and cell bodies of neurons ( I ). J–L , Neurons expressing R5L tau showed an association between tau and PP1 that was similar to WT tau cells and localized to neuronal processes ( K ) and cell bodies ( L ). M–O , Primary antibody delete controls included omission of either Tau7 ( M ), PP1 ( N ), or both ( O ). Primary delete controls showed little to no nonspecific background signal. P–R , PLA in untransduced neurons (No LV) demonstrated an association between endogenous rat tau and PP1, which were evident in neuronal processes ( Q ) and cell bodies ( R ) of neurons. Scale bars: 20 μm for all images.

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Cell Culture, Staining, Recombinant, Western Blot, Isolation, Expressing, Comparison

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: PP1γ knockdown rescues the effects of P301L and R5L on fast axonal transport pause frequency in primary neurons. PP1 expression was knocked down using isoform-specific shRNAs. The shRNAs were validated using a Renilla -Firefly Luciferase assay in HEK293T cells. Renilla -PP1 fusion constructs were cotransfected with shRNAs targeting Renilla (positive control), a nontargeting shRNA sequence (Control shRNA; used as a negative control), or specific PP1 isoforms. Expression knockdown is reported as percentage of an empty shRNA vector control. A , PP1α shRNA produced 87% knockdown from control levels. B , PP1γ shRNA induced 90% knockdown from control levels. Data were compared ( A–B ) using a one-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent replicate ( n = 3; data are mean ± SD; * p ≤ 0.05). C , Neuronal mRNA knockdown was validated using lentiviral expression of PP1α-shRNA, PP1γ-shRNA, or the control shRNA. PP1α and PP1γ mRNA concentrations were determined using ddPCR and normalized to rplp0 reference gene expression. PP1α shRNA treatment knocked down PP1α mRNA compared with control shRNA treatment, whereas PP1γ shRNA treatment only minimally reduced it. D , PP1γ shRNA knocked down PP1γ mRNA compared with control shRNA treatment, whereas PP1α shRNA slightly reduced it. Error bars in C and D represent the 95% confidence intervals. E , Transport experiments were repeated with PP1α, PP1γ-, or control shRNA. Anterograde segment velocity was not significantly different among any of the conditions. Dotted lines represent the baseline values (i.e., the mean of values in the WT tau + control shRNA treatment condition for E and F . F , Retrograde segment velocity was slightly higher with R5L expression compared with WT tau with control shRNA. G , Expressing P301L tau or R5L tau increased total pause frequency compared with WT tau when treated with control or PP1α-shRNA. In contrast, PP1γ-shRNA treatment rescued P301L- and R5L-induced increases in pause frequency by reducing total pause frequency to WT tau control levels. H , P301L and R5L tau increased anterograde pause frequency compared with WT tau + control or PP1α-shRNA. Treatment with PP1γ-shRNA reduced anterograde pause frequency in P301L and R5L neurons compared with WT tau neurons. I , P301L and R5L expression increased retrograde pause frequency compared with WT tau with control shRNA treatments. Coexpression of WT, P301L, or R5L tau with PP1α-shRNA were not different likely because of the enhanced pause frequency in WT. With P301L and R5L, PP1γ-shRNA reduced retrograde pause frequency to levels similar to WT with PP1γ-shRNA and to baseline levels (i.e., WT with control shRNA). Data were compared ( E–I ) using a two-way ANOVA with Tukey's multiple comparison test to compare the three tau groups within each individual shRNA treatment group, and each data point represents an independent replicate ( n = 6; the data are mean ± SD; * p ≤ 0.05; , mean ± SD; , for statistical p values).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Knockdown, Expressing, Luciferase, Construct, Positive Control, shRNA, Sequencing, Control, Negative Control, Plasmid Preparation, Produced, Comparison, Gene Expression

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: FTLD mutant tau enhances the levels of active PP1 in cells and primary neurons. A , Immunoblotting confirmed expression of GFP, WT, P301L, and R5L tau in HEK293T cells coexpressing PP1α. B , Quantification of tau bands indicated similar levels of tau across the three constructs (WT tau, 4.87 ± 0.36; P301L, 5.73 ± 0.58; R5L: 5.67 ± 0.66). C , Immunoblots of total PP1 (green, HaloTag antibody) and inactive PP1 (red, phospho-Thr320 PP1 antibody). D , Quantification of PP1 blots showed that P301L tau increased levels of active PP1α (as indicated by reduced inactive pT320 PP1 signal) compared with GFP control (GFP, 0.731 ± 0.331; WT tau, 0.627 ± 0.054; P301L, 0.388 ± 0.066; R5L, 0.409 ± 0.121). E , Immunoblots of GFP and tau in cell lysates coexpressing PP1β. F , Quantification of tau bands indicated similar expression of tau (WT tau, 7.58 ± 1.66; P301L, 7.94 ± 1.15; R5L, 9.72 ± 3.72). G , Immunoblots of total PP1 (green) and inactive pT316 PP1β (red). H , Quantification of PP1 blots showed that expression of any of the three forms of tau significantly increased active PP1β levels (i.e., reduced inactive pT316 PP1β) compared with GFP control (GFP, 0.0155 ± 0.0017; WT tau, 0.0123 ± 0.0019; P301L, 0.0084 ± 0.0013; R5L, 0.0065 ± 0.0003). P301L and R5L tau significantly increased active PP1β compared with WT tau as well. I , Immunoblots of cells coexpressing GFP, WT tau, P301L tau, or R5L tau and PP1γ. J , Quantification of tau bands showed similar tau expression levels (WT tau, 13.4 ± 3.3; P301L, 15.9 ± 3.4; R5L, 21.6 ± 11.8). K , Immunoblots of total PP1 (green) and inactive pT311 PP1γ (red). L , Quantification of PP1 blots demonstrated that expression of all forms of tau increased active PP1γ (i.e., reduced inactive pT311 PP1γ; GFP, 0.0453 ± 0.0142; WT tau, 0.0161 ± 0.0030; P301L, 0.0045 ± 0.0013; R5L, 0.0065 ± 0.0026). M , Immunoblot of lysates from rat primary neurons expressing GFP or tau (WT, P301L, or R5L) via lentiviral transduction probed with tau (green) and GFP (red) and a loading control (GAPDH). N , Quantification of the exogenous tau bands (top, green) indicated similar levels of tau across the three constructs (WT tau, 19.2 ± 3.9; P301L, 14.8 ± 2.6; R5L, 17.4 ± 4.6). O , Immunoblot of total PP1 (green, PP1 antibody) and inactive PP1 (red, pThr320 PP1 antibody). P , Quantification of the immunoblots demonstrated that P301L tau and R5L tau expression increased levels of active PP1 (i.e., reduced inactive pT320 PP1) compared with a control GFP expression, whereas WT tau did not significantly change active PP1 levels (GFP, 0.0192 ± 0.0012; WT tau, 0.0190 ± 0.0029; P301L, 0.0159 ± 0.0006; R5L, 0.0162 ± 0.0015). Note that tau signal was normalized to GAPDH loading control ( B , F , J , N ), and pT320 PP1 signal was normalized to total PP1 signal ( D , H , L , P ). Data in figure legend are reported in ratios of the signal intensities in arbitrary units. All data are mean ± SD and were compared using repeated measures one-way ANOVAs with a Geisser–Greenhouse correction and Tukey's post hoc multiple comparison test, * p ≤ 0.05).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Mutagenesis, Western Blot, Expressing, Construct, Control, Transduction, Comparison

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: Tau-induced increases in active PP1 is dependent on PAD. PAD, a conformationally displayed biologically active motif in the extreme amino terminus of tau, was deleted in WT and P301L tau by removing amino acids 2–18 (Δ2–18). A , B , The full-length and Δ2–18 constructs were expressed in HEK293 cells, in addition to PP1α, at similar levels ( B ; WT tau, 4.02 ± 0.54; Δ2–18 WT, 3.24 ± 0.63; P301L, 5.32 ± 1.52; Δ2–18 P301L, 4.04 ± 0.58). C , Levels of active PP1α (as indicated by changes in inactive pT320 PP1) were identified using a phospho-Thr320 PP1 antibody (red), and total levels were detected using an anti-HaloTag antibody (green). D , Deletion of PAD resulted in significantly lower levels of active PP1 compared with expression of full-length tau in both WT and P301L backgrounds (WT tau, 0.3409 ± 0.0366; Δ2–18 WT, 0.4232 ± 0.0336; P301L, 0.2147 ± 0.0507; Δ2–18 P301L, 0.4062 ± 0.0406). E , F , WT tau, Δ2–18 WT, P301L, and Δ2–18 P301L were expressed at similar levels ( F ) along with PP1β (WT tau, 1.71 ± 0.14; Δ2–18 WT, 1.64 ± 0.28; P301L, 1.68 ± 0.24; Δ2–18 P301L, 1.78 ± 0.41). G , Immunoblotting of total PP1 (green) and inactive PP1β (red, pT320 PP1 antibody corresponds to pT316 in PP1β). H , Active PP1β levels decreased in the presence of Δ2–18 WT and Δ2–18 P301L compared with full-length WT and P301L tau (WT tau, 0.0393 ± 0.0067; Δ2–18 WT, 0.1525 ± 0.0281; P301L, 0.0162 ± 0.0062; Δ2–18 P301L, 0.1231 ± 0.0125). I , WT tau, Δ2–18 WT, P301L, and Δ2–18 P301L in cells coexpressing PP1γ. J , Quantification indicated similar levels of tau expression with all constructs (WT tau, 8.82 ± 1.27; Δ2–18 WT, 7.26 ± 1.57; P301L, 8.32 ± 2.32; Δ2–18 P301L, 6.96 ± 1.73). K , Immunoblots of total PP1 (green) and inactive PP1γ (red, pT320 PP1 antibody corresponds to pT311 in PP1γ). L , The Δ2–18 WT resulted in decreased levels of active PP1γ compared with full-length counterparts (WT tau, 0.0578 ± 0.0099; Δ2–18 WT, 0.1428 ± 0.0154; P301L, 0.0101 ± 0.0022; Δ2–18 P301L, 0.0862 ± 0.0471). P301L tau expression led to higher levels of active PP1 compared with WT tau, and this effect was reduced about ninefold on PAD deletion in Δ2–18 P301L. Tau signal was normalized to GAPDH loading control ( B , F , J ) and pT320 PP1 signal was normalized to total PP1 signal ( D , H , L ). Data in legend are reported in ratios of the signal intensities in arbitrary units. All data in figure are mean ± SD and were compared using repeated measures one-way ANOVAs with a Geisser–Greenhouse correction and Tukey's post hoc multiple comparison test, * p ≤ 0.05).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Construct, Expressing, Western Blot, Control, Comparison

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: P301L tau increases the association with PP1γ isoform in primary hippocampal neurons. A , PLA experiments were repeated with the Tau7 antibody and a PP1γ-specific antibody verified with immunoblot. Recombinant PP1 isoforms were probed with a PP1γ antibody (green) and a 6xHis antibody (red). B , PLA puncta were quantified and normalized to area of Tau12 staining (i.e., exogenous tau staining). P301L tau (28.0 ± 7.0 puncta/nm 2 ) expression increased levels of PLA signal compared with WT tau (11.3 ± 1.9 puncta/nm 2 ), whereas R5L (16.2 ± 2.1 puncta/nm 2 ) expression did not significantly change. Data represent mean ± SD of three replicates and were analyzed using a repeated measures one-way ANOVA and Tukey's post hoc multiple comparison test; * p ≤ 0.05. C , A Tau7 primary antibody deletion demonstrated the lack of background signal when PLA is performed with the PP1γ antibody only. D–F , Associations between WT tau and endogenous rat PP1γ (PLA, green puncta; Tau12, red) were identified in the neuronal processes ( E ) and cell bodies ( F ) of the neurons. G – I , Lentiviral expression of P301L tau increased PLA puncta, which were found in processes ( H ) and cell bodies of neurons ( I ). J–L , PLA in the presence of R5L tau also demonstrated an association between tau and PP1γ, localized to the processes ( K ) and cell bodies ( L ) of the neurons. M–O , PLA was performed in untransduced neurons to identify an association between endogenous rat tau and PP1γ, also found in neuronal processes ( N ) and cell bodies ( O ). Scale bars: 20 μm for all images.

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Western Blot, Recombinant, Staining, Expressing, Comparison

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: P301L mutation in tau enhances the interaction with PP1α and γ isoforms in pull-down (PD) assays. A , Schematic showing assay design. Individual tau constructs were coexpressed with HaloTag-PP1 (left) or HaloTag-only controls (right) in HEK293T cells. Lysates prepared from these cells were incubated with HaloLink resin, which covalently binds to HaloTag. We quantified the amount of tau signal (pan-tau R1 antibody, green) eluted from each pull down by immunoblot (PD elution) and normalized to the reduction in Halo signal (anti-Halo antibody, red) from prebinding to postbinding lysate samples. B , WT, P301L tau, and R5L tau proteins were detected in the elution samples after a pull down with Halo-PP1α (left, red box). Tau was not detected in the HaloTag-only control (right, red box). Lysate samples obtained before (Input) and after pull down with the HaloLink resin (Post PD) were probed to confirm similar protein expression and HaloTag binding to resin. C , Quantitation of tau in the elution samples indicate that P301L tau (0.673 ± 0.429) significantly increases the interaction with PP1α when compared with WT (0.160 ± 0.147) and R5L tau (0.107 ± 0.041) and the control with HaloTag-only (WT tau, 0.004 ± 0.002; P301L, 0.003 ± 0.003; R5L, 0.003 ± 0.001). D , A relatively low level of WT and P301L tau was eluted from the Halo-PP1β pull down but not the HaloTag-only control. The pre- and post-pull-down lysate samples from the Halo-PP1β and Halo-only lysates confirm similar expression and pull-down efficiency. E , Quantitation of tau in the pull-down elution samples indicate that P301L tau (0.061 ± 0.038) significantly increases the interaction with PP1β when compared with the P301L + HaloTag control (−0.001 ± 0.001). Notably, the amount of tau in the elution with Halo-PP1β pull down is much lower for all forms (WT tau, 0.023 ± 0.024; R5L, 0.009 ± 0.005) and not significantly higher than the HaloTag-only controls (WT tau, 0.001 ± 0.001; R5L, −0.001 ± 0.001). F , WT and P301L tau proteins were detected in the elution samples after a pull down with Halo-PP1γ. The pre- and post-pull-down lysate samples from the Halo-PP1γ and Halo-only lysates confirm similar expression and pull-down efficiency. G , Quantitation of tau in the elution samples indicate that P301L tau (0.540 ± 0.209) significantly increases the interaction with PP1γ when compared with WT tau (0.125 ± 0.069), R5L tau (0.111 ± 0.075), and the P301L + HaloTag control (0.000 ± 0.001). Tau was also absent in HaloTag pull-down controls (WT tau, −0.001 ± 0.002; R5L, 0.001 ± 0.002). All data in the legend are reported as ratios of the signal intensities in arbitrary units. Statistical comparisons were performed using a repeated measures two-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent experimental replicate ( n = 4 independent replicates; data are mean ± SD; * p ≤ 0.05 for comparisons among the tau group; # p ≤ 0.05 for the comparison between Halo-PP1 and Halo-only pull downs).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Mutagenesis, Construct, Incubation, Western Blot, Control, Expressing, Binding Assay, Quantitation Assay, Comparison

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: Tau protein interacts with PP1α and PP1γ, and the P301L tau mutation enhances the interaction in intracellular nBRET assay. A , Tau proteins were tagged with a luciferase enzyme (NanoLuc) and coexpressed in HEK293T cells in the presence of an individual Halo-PP1 isoform or HaloTag-only control protein. A fluorescent ligand binds to the HaloTag, forming the acceptor molecule. Light emitted from the luciferase (donor) activates the fluorophore (acceptor) if they are within ∼37 Å of each other because of a specific protein–protein interaction. The donor and acceptor signals were measured using filtered luminescence and reported as the nBRET ratio. The acceptor:donor DNA ratio was varied to generate a donor saturation assay curve. B , The hyperbolic shape of the donor saturation curves indicated a specific protein–protein interaction between Halo-PP1α and WT tau (solid black), P301L tau (solid gray), and R5L tau (dashed black) tagged with NanoLuciferase. C , A strong interaction was not detected for the same forms of tau in the presence of Halo-PP1β. D , Each form of tau displayed a clear interaction with Halo-PP1γ. E , There was no evidence of a specific interaction between the Halo-only control and any of the tau proteins. F , The nBRET was performed at an acceptor-to-donor DNA ratio of 100:1 with four independent replicates to compare nBRET ratios among the tau groups with Halo-PP1 or Halo-only. Each tau displayed significantly higher nBRET ratios with Halo-PP1α (WT tau, 9.57 ± 1.05 mBU; P301L, 20.40 ± 2.58 mBU; R5L, 10.25 ± 0.57 mBU) compared with Halo-only controls (WT tau, 5.168 ± 0.16 mBU; P301L, 5.814 ± 0.36 mBU; R5L, 5.418 ± 0.30 mBU). P301L tau nBRET ratios were significantly higher than WT and R5L tau with Halo-PP1α. G , There was no significant increase detected for any of the individual tau constructs with Halo-PP1β (WT tau, 6.067 ± 1.69 mBU; P301L, 8.323 ± 2.59 mBU; R5L, 6.325 ± 1.53 mBU) compared with Halo-only or among the tau constructs and Halo-PP1β. H , The nBRET ratios for each of the tau forms were significantly higher with Halo-PP1γ (WT tau, 9.579 ± 0.429 ± 1.71 mBU; P301L, 20.38 ± 1.99 mBU; R5L, 8.939 ± 1.37 mBU) compared with Halo-only controls. The nBRET ratios between Halo-PP1γ and P301L tau significantly increased compared with WT and R5L tau. Statistical comparisons for F–H were performed using a two-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent experimental replicate (data are mean ± SD; * p ≤ 0.05 for the comparison among the tau groups; # p ≤ 0.05 for the comparison between Halo-PP1 and Halo-only groups).

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: Mutagenesis, Luciferase, Control, Saturation Assay, Construct, Comparison

Journal: The Journal of Neuroscience

Article Title: Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism

doi: 10.1523/JNEUROSCI.1914-20.2021

Figure Lengend Snippet: Quantitative data for tau + shRNA axonal transport experiments

Article Snippet: Blots were probed for phospho-PP1 (pT320; 1:500; catalog #2581, Cell Signaling Technology; RRID: AB_330823 ), HaloTag (1:1000; catalog #G9211, Promega; RRID: AB_2688011 ), Total PP1 (E-9; 1:400; catalog #sc-7482, Santa Cruz Biotechnology; RRID: AB_628177 ), GAPDH (loading control; 1:2000; catalog #5174, Cell Signaling Technology; RRID: AB_10622025 ), Tau5 (10 ng/ml; catalog# Tau5, Nicholas M. Kanaan at Michigan State University; RRID: AB_2721194 ; ; ), and GFP (1:30,000; catalog #ab290, Abcam; RRID: AB_303395 ).

Techniques: shRNA, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Pathological Tau From Alzheimer’s Brain Induces Site-Specific Hyperphosphorylation and SDS- and Reducing Agent-Resistant Aggregation of Tau in vivo

doi: 10.3389/fnagi.2019.00034

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: PP1 , Poly- , R , Total PP1 , Santa-Cruz (sc-7482/1032).

Techniques: Transduction

Journal: Frontiers in Aging Neuroscience

Article Title: Pathological Tau From Alzheimer’s Brain Induces Site-Specific Hyperphosphorylation and SDS- and Reducing Agent-Resistant Aggregation of Tau in vivo

doi: 10.3389/fnagi.2019.00034

Figure Lengend Snippet: Expression of tau phosphatases in AD P-tau-injected hippocampus. PP1, protein phosphatase 2A (PP2A), demethylated PP2A, PP2B and PP5 in the ipsilateral hippocampus in Tg/hTau and Tau−/− mice injected with AD P-tau or saline were analyzed by Western blots developed with antibodies indicated (A) . The levels of demethylated PP2A and PP2A (B) and PP1, PP2B and PP5 (C) were statistically analyzed with two-way ANOVA post Sidak’s multiple comparisons test after normalized with PP2A (for DM-PP2A) or GAPDH (for PPs) and are presented as mean ± SD. **** p < 0.0001.

Article Snippet: PP1 , Poly- , R , Total PP1 , Santa-Cruz (sc-7482/1032).

Techniques: Expressing, Injection, Saline, Western Blot