recombinant human pten  (R&D Systems)

Journal: Neuron

Article Title: Tau reduction prevents key features of autism in mouse models

doi: 10.1016/j.neuron.2020.01.038

Figure Lengend Snippet: (A–B) Colocalization of tau and PTEN in primary cortical neurons analyzed by PLA. (A) WT rat neurons (DIV 10) were labeled with the tau antibody Tau5 (top), a PTEN antibody (middle), or both (bottom). The PLA signal (red) indicates close proximity of the antigens. Neurons were counterstained with DAPI (blue) and an antibody to MAP2 (green). Scale bar: 30 μm. (B) Mapt−/− mouse neurons were transduced on DIV 7 with lentiviral vectors encoding GFP alone (top) or GFP and 0N4R mouse tau (bottom), labeled with antibodies against tau (Tau5) and PTEN, and subjected to PLA on DIV 10. Scale bar: 50 μm. Some of the PLA signals in (A) and (B) appear to reside outside of cells because of weak MAP2 or GFP staining of the fine neuritic processes with which they are associated. (C) Lipid phosphatase activity of PTEN measured under cell-free conditions in the presence of different recombinant human tau species. Albumin was used as the negative control (Control). n = 3 independent experiments, each including two to three replicates per condition. To combine data from independent experiments, measurements in (C) were normalized to the mean PIP2 concentration at 1 ng/μl PTEN with albumin (defined as 1.0). (D–H) Analysis of interactions between hTau and hPTEN in transiently transfected HEK-293 cells. (D) Schematic of WT 1N4R hTau and deletion mutants lacking the indicated domains. Numbers indicate amino acid positions in 1N4R hTau and those in parentheses in 2N4R hTau. (E) Western blot analysis of immunoprecipitates (left) and column flow through (right) from cells expressing GFP-P2A alone (Ctr) or the constructs in (D), after immunoprecipitation of cell lysates with an antibody against PTEN. Blots were probed with antibodies to tau (Tau46 plus Tau5) and PTEN. Similar results were obtained in two additional experiments (not shown). (F and G) BiFC assay of HEK-293 cells expressing the constructs indicated at the top in (F) and shown in (G). WT human PTEN (hPTEN: PBD, PIP2 binding domain; PTP, protein tyrosine phosphatase domain; CT, C-tail domain) was tagged with VN, and WT 2N4R hTau and its mutants were tagged with VC. Numbers indicate amino acid positions in hPTEN and 2N4R hTau. hPTEN and VC were detected by immunocytochemistry (ICC). Interactions between VN-hPTEN and hTau-VC were detected by Venus fluorescence resulting from close proximity between VN and VC. Scale bar: 40 μm. (H) Quantitation of BiFC-Venus signals normalized to VC immunoreactivity. n = 3 independent experiments, each including two to three coverslips/condition. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. PTEN concentration-matched control (C) or VC alone (H), determined by multiple Welch’s t tests (C) or one-way ANOVA with Holm-Sidak test (H). n.s., not significant. Values are means ± SEM.

Article Snippet: Briefly, recombinant WT human PTEN (0.125–1.0 ng/μl; Echelon Biosciences, E-3000) was incubated with matching concentrations (in weight/volume) of recombinant 0N3R, 0N4R, 1N4R, or 2N4R hTau (Tau-352, Tau-383, Tau-412, or Tau-441, respectively; rPeptide) or with protease-free albumin (Gemini Bio-Products, 700–101P) as a negative control, in PTEN Reaction Buffer (K-4704, Echelon Biosciences) with 10 mM dithiothreitol (K-DTT1, Echelon Biosciences) containing PIP3 substrate (8 μM, K-4702; Echelon Biosciences) for 1–2 hours at 37° C in low-bind, 0.

Techniques: Labeling, Staining, Activity Assay, Recombinant, Negative Control, Concentration Assay, Transfection, Western Blot, Expressing, Construct, Immunoprecipitation, Bimolecular Fluorescence Complementation Assay, Binding Assay, Immunocytochemistry, Fluorescence, Quantitation Assay

Journal: Nature communications

Article Title: Hyperphosphorylated PTEN exerts oncogenic properties.

doi: 10.1038/s41467-023-38740-x

Figure Lengend Snippet: Fig. 2 | Tumor protection in PtenS380A/A and PtenΔ4/Δ4 mice despite AKT signaling. a Overall and (b) tumor-specific incidence of tumors at 9 months in FVB mice of indicated genotypes. Number of mice (n) for (a–c) is indicated in (a). Incidence of specific tumorsis basedon macroscopic screening. “Other tumors” include lipomas and tumors in breast, uterus and liver. Photos above graph show examples of below graphed tumors. c Incidence at 9 months of lymphadenopathy. We note that the incidence in +/+ was 0%. Photo depicts an example of lymphadenopathy in a sub- mandibular lymph node of a Pten+/– mouse. d, e As (a, b) but now at 16 months,

Article Snippet: In vitro protein binding studies were conducted in 100 μl PBS at RT using recombinant human PTEN (R&D systems Cat. #847-PN) and recombinant human β-catenin (Sino Biological Cat. #11279-H20B).

Techniques:

Journal: Nature communications

Article Title: Hyperphosphorylated PTEN exerts oncogenic properties.

doi: 10.1038/s41467-023-38740-x

Figure Lengend Snippet: Fig. 3 | PtenHNP/HNP males have a less advanced PIN phenotype than Pten+/– males despite similarly reduced PTEN levels. a CRISPR-CAS9-targeting design of Pten hypomorphic mice. PtenHN, hypomorph Neo; PtenHNP, hypomorph Neo & PolyA. b WB of prostate lysates harvested at 2 months, probed for PTEN and activation of AKT signaling pathway. Blot is representative of at least 3 individual samples. Ponceau S (PonS) staining served as loading control. c Quantitation of PTEN level in

Article Snippet: In vitro protein binding studies were conducted in 100 μl PBS at RT using recombinant human PTEN (R&D systems Cat. #847-PN) and recombinant human β-catenin (Sino Biological Cat. #11279-H20B).

Techniques: CRISPR, Activation Assay, Staining, Control, Quantitation Assay

Journal: Nature communications

Article Title: Hyperphosphorylated PTEN exerts oncogenic properties.

doi: 10.1038/s41467-023-38740-x

Figure Lengend Snippet: Fig. 4 | PIN lesion formation in Pten+/– males is characterized by loss of PTEN catalytic function. a Immunostaining of consecutive sections of normal and PIN lesion (*) tubules of indicated genotypes stained for H&E, PTEN and P-AKTS473. Scale bar is 100 µm. b Close-up of a small lesion of epithelial cells that have lost PTEN expression and have gained P-AKTS473 expression. c Quantitation of PIN lesions with increase in P-AKTS473 expression and simultaneous loss of PTEN. 5 mice per geno- type with 12-55 PIN lesions per mouse were analyzed by IF staining. Data are

Article Snippet: In vitro protein binding studies were conducted in 100 μl PBS at RT using recombinant human PTEN (R&D systems Cat. #847-PN) and recombinant human β-catenin (Sino Biological Cat. #11279-H20B).

Techniques: Immunostaining, Staining, Expressing, Quantitation Assay

Journal: Nature communications

Article Title: Hyperphosphorylated PTEN exerts oncogenic properties.

doi: 10.1038/s41467-023-38740-x

Figure Lengend Snippet: Fig. 7 | Hyperactive WNT signaling predisposes PtenS380D mice to PIN lesions. a Mixtures of recombinant human PTEN and β−catenin proteins subjected to immunoprecipitation with PTEN or corresponding control (anti-RFP) antibodies and analyzed by immunoblotting using PTEN and β−catenin antibodies.The blot is representative of 3 independent experiments. b Immunoblot of 2-month-old frac- tionated prostate lysates of indicated genotypes subjected to immunoprecipitation with PTEN or control antibody and analyzed with the indicated antibodies. Blots represent 3 independent experiments. * Marks IgG band, only visible in the nuclear fraction because of the necessary long exposure to be able to detectthe PTEN band.

Article Snippet: In vitro protein binding studies were conducted in 100 μl PBS at RT using recombinant human PTEN (R&D systems Cat. #847-PN) and recombinant human β-catenin (Sino Biological Cat. #11279-H20B).

Techniques: Recombinant, Immunoprecipitation, Control, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Gut microbiota relieves inflammation in the substantia nigra of chronic Parkinson's disease by protecting the function of dopamine neurons

doi: 10.3892/etm.2021.10974

Figure Lengend Snippet: FMT suppresses inflammation in the substantia nigra in an MPTP-induced Parkinson's disease mouse model. (A) Western blotting bands of Arginase, GSK3β, IL-1β, iNOS, α7n AchR and p-PTEN. Western blotting bands statistical quantification of (B) Arginase, (C) GSK3β, (D) IL-1β, (E) iNOS, (F) p-PTEN and (G) α7n AchR. Gene expression levels of (H) Arginase, (I) GSK3β, (J) IL-1β, (K) iNOS, (L) TNF-α, (M) IL-1β, and (N) TGF-β. n=3. * P<0.05, ** P<0.01, *** P<0.005 and **** P<0.001 vs. control; # P<0.05 and ## P<0.01 vs. MPTP. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; FMT, faecal microbiota transplantation; Iba1, allograft inflammatory factor 1; iNOS, inducible nitric oxide synthase; α7n AchR, α-7 nicotinic acetylcholine receptor; p-, phosphorylated.

Article Snippet: The primary antibodies used were arginase (cat. no. ab233548; 1:5,000; Abcam), GSK3β (cat. no. 21002; 1:200; Signalway Antibody LLC), IL-1β (cat. no. ab5076; 1:1,000; Abcam), iNOS (cat. no. AF0199; 1:2,000; Affinity Biosciences, Ltd.), phosphorylated (p)-PTEN (cat. no. ab109454; 1:10,000; Abcam), PTEN (cat. no. ab267787; 1:1,000; Abcam) and α-7 nicotinic acetylcholine receptor (α7n AChR; cat. no. ab216485; 1:1,000; Abcam).

Techniques: Western Blot, Gene Expression, Control, Transplantation Assay