Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: The C-terminus of calpain-2 binds to the PDZ domains of PTPN13. (A) Overlay of a PDZ domain array with the GST-tagged C-terminus of calpain-2 (800 nM). The GST-calpain-2 C-terminus exhibited specific binding to several PDZ domains, with PDZ domains 3, 4 and 5 of PTPN13 showing the strongest interactions (arrows). This overlay was repeated three times with similar results. ( B ) Schematic illustration of PTPN13 domains, including kinase non-catalytic C-lobe (KIND) domain and Four-point-one, ezrin, radixin, moesin (FERM) domain at the N-terminus, followed by 5 PDZ domains, and a Protein tyrosine phosphatase (PTP) domain at the C-terminus. ( C ) Co-immunostaining of calpain-2 and PTPN13 or calpain-1 and PTPN13 in the dendrites of CA1 pyramidal neurons. Scale bar = 20 µm. ( D ) Quantification of immunostaining results. Calpain-2 and PTPN13 IMF intensity were analyzed and exhibited a higher Pearson’s correlation coefficient, as compared to calpain-1 and PTPN13 IMF in CA1. N = 4 (animals). In each animal, 4–6 CA1 regions were assessed. ***P < 0.001. Two-tailed t -test. ( E ) Co-immunoprecipitation of PTPN13 with calpain-2, tau and c-Abl in P2 homogenates of mouse brain. Calpain-2 C-terminal peptide (C2CP) was added to the homogenates at a final concentration of 10 µM and incubated for 1 h before co-IP. Immunoprecipitation with rabbit IgG was performed as a negative control. Full-length blots are presented in Supplementary Figure . ( F ) Quantification of Western blots. Application of calpain-2 C-terminal peptide significantly reduced co-IP of PTPN13 with calpain-2. Data are means ± SEM of 3 independent experiments. **p < 0.01. Two-tailed t -test.

Article Snippet: The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich).

Techniques: Binding Assay, Immunostaining, Two Tailed Test, Immunoprecipitation, Concentration Assay, Incubation, Co-Immunoprecipitation Assay, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: Calpain-2 truncates PTPN13, generating stable breakdown products. ( A ) P2 fractions prepared from mouse brain homogenates were treated with 20 µM or 2 mM Ca 2+ and incubated for 20 or 60 min at 37 °C. In some groups, a calpain-2 selective inhibitor NA101 (200 nM) was added 10 min before Ca 2+ treatment. The truncation of PTPN13 and spectrin was analyzed using WB. Full-length blots are presented in Supplementary Figure . ( B ) Illustration of the two potential calpain cleavage sites on PTPN13 identified by calpain cleavage site predictor (SVM RBF prediction model). The cleavage at 1488 is predicted to generate a 170 kDa N-terminal fragment and the cleavage at 1965 to generate a 210 kDa N-terminal fragment. These two fragments contain the epitope of PTPN13 antibody (aa1279–1883, as indicated in the antibody manual) used in the WB of panel A and should be detected by this antibody. According to our result, the actual epitope of this antibody should be within aa1279–1488.

Article Snippet: The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich).

Techniques: Incubation

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: Calpain-2 mediated cleavage of PTPN13 regulates tyrosine phosphorylation of c-Abl and tau. ( A ) Cos-1 cells were transfected with GFP-Tau and c-Fyn (lane 1), GFP-Tau and Flag-SYK (lane 2) or GFP-Tau plus Flag-His-c-Abl (lane 3). GFP-Tau was immunoprecipitated using anti-GFP antibody. Tyrosine phosphorylation of GFP-Tau was examined using anti-phosphotyrosine antibody. Full-length blots are presented in Supplementary Figure . ( B ) Cos-1 cells were transfected with GFP-Tau plus Flag-His-Abl (lane 1), GFP-Tau plus Flag-His-Abl plus Flag-PTPN13 WT (lane 2) and GFP-Tau plus Flag-His-Abl plus Flag-PTPN13 D2359A (lane 3). Flag-PTPN13 D2359A is an inactive mutant of PTPN13. GFP-Tau was immunoprecipitated by anti-GFP antibody and was precipitated GFP-Tau was analyzed with an anti-phosphotyrosine antibody. Full-length blots are presented in Supplementary Figure . ( C ) Cos-1 cells were transfected with GFP-Tau plus c-Fyn (lane 1), GFP-Tau plus c-Fyn plus Flag-PTPN13 WT (lane 2) and GFP-Tau plus c-Fyn plus Flag-PTPN13 D2359A (lane 3). Tyrosine phosphorylation of GFP-Tau was examined. Full-length blots are presented in Supplementary Figure . ( D ) Cos-1 cells were transfected with Flag-c-Abl (lane 1), Flag-c-Abl plus Flag-PTPN13 WT (lane 2) and Flag-c-Abl plus Flag-PTPN13 D2359A (lane 3). Flag-c-Abl was pulled-down using anti-Abl antibody. Tyrosine phosphorylation of Abl was examined using anti-phosphotyrosine, phospho-c-Abl Tyr245 and phospho-c-Abl Tyr412 antibodies. Full-length blots are presented in Supplementary Figure . ( E ) NMDA treatment of acute hippocampal slices from calpain-1 KO mice at postnatal day 10. Slices were treated with 10 µM NMDA at 37 °C for 30 min. In one group, slices were pre-treated with a calpain inhibitor (calpain inhibitor III, 10 µM) for 10 min before adding NMDA. Levels of indicated proteins in the collected slices were analyzed by WB. Lane 1 and 2 are two replicates. Full-length blots are presented in Supplementary Figure . ( F ) Quantification of WB. Ratios of P13BPs to full-length PTPN13 and phospho-c-Abl Tyr245 to total c-Abl were significantly increased with NMDA treatment. Treatment with CI-III significantly inhibited NMDA-mediated changes. Data are means ± SEM of 4 independent experiments. *p < 0.05, **p < 0.01. Two-way ANOVA followed by Bonferroni test.

Article Snippet: The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich).

Techniques: Transfection, Immunoprecipitation, Mutagenesis

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: TBI triggers calpain-2 mediated PTPN13 cleavage and tyrosine phosphorylation of c-Abl and tau. ( A ) A calpain-2 selective inhibitor inhibits TBI-triggered PTPN13 cleavage and tyrosine phosphorylation of c-Abl and tau. Ipsilateral cortex of WT mice was collected and homogenized 6 h after surgery. C-Abl or tau was then immunoprecipitated from homogenates to test its tyrosine phosphorylation. Lane 1, Sham surgery. Immunoprecipitation was performed with tau or c-Abl antibody. Lane 2, Controlled cortical impact (CCI). Immunoprecipitation was performed with tau or c-Abl antibody. Lane 3, NA101 was injected i.p. 1 h after CCI. Immunoprecipitation was performed with tau or c-Abl antibody. Lane 4, Sham surgery. Immunoprecipitation was performed with mouse or rabbit IgG as a negative control. Full-length blots are presented in Supplementary Figure . ( B ) Ipsilateral cortex of calpain-1 KO mice was collected and homogenized 6 h after CCI or sham surgery. PTPN13 and spectrin cleavage and tau tyrosine phosphorylation were analyzed with WB. Lane 1, Sham surgery. Immunoprecipitation was performed with tau antibody. Lane 2, CCI. Immunoprecipitation was performed with tau antibody. Lane 3, Sham surgery. Immunoprecipitation was performed with mouse IgG as a negative control. Full-length blots are presented in Supplementary Figure . ( C ) Quantification of WB results similar to panel A. The ratios of P13BPs to PTPN13, phospho-tyrosine tau to total tau, and phospho-Tyr245 of c-Abl to total c-Abl were significantly increased following CCI. Injection with NA101 significantly inhibited those changes following CCI. Data are means ± SEM of 3–5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA followed by Bonferroni test. ( D ) Quantification of WB results similar to panel B. The ratios of P13BPs to PTPN13, and phospho-tyrosine tau to total tau were significantly increased following CCI. Data are means ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01. Two-way ANOVA followed by Bonferroni test. ( E – G ) IHC with a phospho-c-Abl (Tyr245) antibody in coronal brain sections (Bregma −0.58 mm) 6 h after TBI. Vehicle (5% DMSO in PBS) or NA101 (0.3 mg/kg) was injected i.p. 1 h after TBI. Scale bar in the DAPI images under low-magnification, 500 µm. Scale bar in the zoomed-in images, 100 µm. ( H ) High-magnification images of phospho-c-Abl (Tyr245) staining of cortical neurons in ipsilateral cortex 6 h after TBI. Scale bar, 10 µm. ( I ) Quantification of IHC results. Three coronal brain sections (Bregma −0.58 mm, −1.58 mm and −1.94 mm) from each brain were immunostained and IMF intensity analyzed. Changes in phospho-Tyr245 level were calculated as the mean fluorescence intensity (MFI) of p-Tyr245 staining in the ipsilateral side of the insult subtracted by the MFI of p-Tyr245 staining in the contralateral side. Changes in p-Tyr245 in all 3 sections from the same brain were averaged. N = 3–5 (animals). *p < 0.05, **p < 0.01. One-way ANOVA followed by Bonferroni test.

Article Snippet: The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich).

Techniques: Immunoprecipitation, Injection, Negative Control, Staining, Fluorescence

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: Inhibition of calpain-2 or c-Abl attenuates the formation of tau oligomers in brain following TBI. (A) IHC with an anti-tau oligomer antibody (T22) in cortical and hippocampal CA1 areas of coronal brain sections 24 h after TBI or sham surgery. Vehicle (5% DMSO in PBS), NA101 (0.3 mg/kg) or Nilotinib (25 mg/kg) was injected i.p. 1 h after TBI. Scale bar = 100 µm. ( B ) Quantification of IHC results. Three coronal brain sections (Bregma −1.58 mm, −1.94 mm and −2.30 mm) from each brain were imaged. MFIs in cortical and CA1 areas from each section were measured in ImageJ. N = 3 for Sham, C2I and Nilotinib. N = 5 for Vehicle. **p < 0.01, ****p < 0.0001 Sham vs. Vehicle, # p < 0.05 C2I vs. Vehicle, ## p < 0.01 Nilotinib vs. Vehicle. Two-way ANOVA followed by Bonferroni test. ( C ) ELISA analysis of brain homogenates using Tau-5 and T22 antibodies 24 h after TBI or sham surgery. Vehicle (5% DMSO in PBS), NA101 (0.3 mg/kg) or Nilotinib (25 mg/kg) was injected i.p. 1 h after TBI. The ratios of tau oligomers to total tau were analyzed. N = 4 (animals). ***p < 0.001 sham vs. vehicle, # p < 0.05 vehicle vs. C2I. ## p < 0.01 vehicle vs. Nilotinib. One-way ANOVA followed by Bonferroni test. ( D ) Levels of Tau monomers and oligomers and Serine/Threonine phosphorylation of tau at S202, T205, T231 and S416 in ipsilateral cortical homogenates collected 24 h after sham (lane 1) or TBI (lane 2) or TBI plus NA101 injection (lane 3) were analyzed with WB of non-reducing SDS-PAGE (NuPAGE 4–12% Bis-Tris gel). Full-length blots are presented in Supplementary Figure . ( E ) Quantification of tau oligomers (boxed area) as detected by WB. N = 3 (animals). ****p < 0.0001 sham vs. TBI. ### p < 0.001 TBI vs. TBI + NA101. Two-way ANOVA followed by Bonferroni test. ( F ) Post-mortem hippocampal samples from 4 AD patients and 4 age-matched controls (N) were probed with antibodies against PTPN13 and actin in WB. Full-length PTPN13 at 275 kDa and a P13BP at approximately 210 kDa were detected. ( G ) Quantification of the Western blots. The ratio of P13BP to full-length PTPN13 was significantly higher in AD patients than that in controls. N = 4. *p < 0.05. Two-tailed t -test.

Article Snippet: The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich).

Techniques: Inhibition, Injection, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Two Tailed Test

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: A new link between TBI, calpain-2, tauopathy and AD pathogenesis. TBI triggers calpain-2 activation. PTPN13 is cleaved and inactivated. This results in increased tyrosine phosphorylation of c-Abl at tyr245, which increases its kinase activity and phosphorylates tau at tyr394. Elevation of tau tyrosine phosphorylation contributes to tau oligomer accumulation, which may lead to increased risk of AD or AD-related disease. Administration of a calpain-2 selective inhibitor post-TBI reduces TBI-induced calpain-2 activation, tau tyrosine phosphorylation and tau oligomer formation, thus may inhibit tauopathy and AD pathogenesis.

Article Snippet: The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich).

Techniques: Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions

doi: 10.1038/srep08530

Figure Lengend Snippet: (A), a region within the C-terminus of Flexi cloned TNIP2 is important for its association with PTPN13. Plasmids expressing the six Halo-tagged constructs indicated were transiently transfected in HEK293T cells for Halo affinity purification followed by MudPIT mass spectrometry analysis. Relative amounts of the five prey proteins indicated in enriched using each of these six baits (FDR < 0.05) are indicated according to their relative dNSAF value. Average prey dNSAF values were calculated from between three and six replicate experiments for each bait (see ). Average prey dNSAF values were then normalized to the average bait dNSAF to generate relative dNSAF values. (B), the association of Flexi-cloned TNIP2 and PTPN13 depends on the C-terminal valine cloning scar. Whole cell extracts from HEK293T cells transfected with the indicated constructs were subjected to Halo affinity chromatography and samples were analysed by SDS-PAGE followed by Western blotting. TNIP2 protein was visualized using rabbit anti-TNIP2 or rabbit anti-PTPN13 primary antibodies, and Alexa-680 labeled anti-rabbit secondary antibodies. Note the change in molecular weight of the TNIP2 bait after purification, which involves removal of the 33 kDa Halo tag. Western blots were imaged using a Li-Cor infra-red imaging system. (C), Halo purified proteins from HEK293T cells transfected with Halo-TNIP2 253–429 (5 biological replicates), Halo-TNIP2 253–429 no valine (3 biological replicates) and Halo-PTPN13 (3 biological replicates) were analysed by mass spectrometry. The numbers of distributed MS/MS spectra corresponding to the proteins TNIP2, PTPN13 and STXBP4 from each of these replicates is indicated. None of these proteins were detected in 9 biological replicates of control purifcations using cells expressing the Halo tag alone.

Article Snippet: Rabbit anti-TNIP2 (NBP1-32689) and rabbit anti-PTPN13 (NB100-56139) polyclonal antibodies were from Novus Biologicals.

Techniques: Clone Assay, Expressing, Construct, Transfection, Affinity Purification, Mass Spectrometry, Cloning, Affinity Chromatography, SDS Page, Western Blot, Labeling, Molecular Weight, Purification, Imaging, Tandem Mass Spectroscopy, Control

Journal: Scientific Reports

Article Title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions

doi: 10.1038/srep08530

Figure Lengend Snippet: Part of the PTPN13 ORF coding for a 903 aa region, which included the five PDZ domains, was subcloned into FLAG-pcDNA5/FRT and coexpressed in HEK293T cells with or without Halo-TNIP2 (with the valine cloning scar) as indicated. Lysates were subjected to Halo affinity purification and the resulting eluates analysed by SDS-PAGE and Western blotting. Proteins were detected using anti-FLAG® M2 mouse monoclonal and anti-TNIP2 rabbit polyclonal primary antibodies, and with IRDye® 680LT labeled anti-mouse (red) and IRDye® 800CW anti-rabbit (green) secondary antibodies. Proteins were visualized using a LI-COR® Odyssey® infrared imaging system.

Article Snippet: Rabbit anti-TNIP2 (NBP1-32689) and rabbit anti-PTPN13 (NB100-56139) polyclonal antibodies were from Novus Biologicals.

Techniques: Cloning, Affinity Purification, SDS Page, Western Blot, Labeling, Imaging