recombinant human shp2  (Absolute Biotech Inc)

Journal: Nature cell biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells.

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: Fig. 5 | ITGB1 is a substrate for PTP-PEST and Shp2. a,b, A malachite green assay for free phosphate release after incubation of phosphorylated/non- phosphorylated ITGB1 peptides with recombinant Shp2 (n = 5 independent replicates, each performed in triplicate) (a) or PTP-PEST (n = 4 independent replicates, each performed in triplicate) (b). The significance was assessed using a Kruskal–Wallis test with a Dunn’s correction for multiple comparisons. The data are presented as the mean ± s.e.m. c,d, Schematics of FRET experiments (left) using mRuby2-tagged ITGB1 and Clover-tagged PTPs. Representative FLIM–FRET images (right) and quantification of apparent FRET efficiency of MM231 cells with stable expression of either ITGB1(WT)–mRuby2 or ITGB1(YYFF)–mRuby2

Article Snippet: Each fragment (2,400 pmol per peptide per reaction) was incubated separately for 1 h at 37 °C with recombinant Shp2 (0.05 μg ml−1; R&D Systems, 1894-SH-100) or PTP-PEST (0.05 μg ml−1; SignalChem, P39-21G-10) in phosphatase buffer (HEPES buffer, pH 7.5 (50 mM)/EDTA (0.2 mM)/DTT (5 mM)/Triton X-100 (0.01%)), before incubation with Malachite Green Reagent (100 μl per reaction).

Techniques: Malachite Green Assay, Incubation, Recombinant, Expressing

Journal: The EMBO Journal

Article Title: PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

doi: 10.15252/embj.2020106113

Figure Lengend Snippet: Transmigration of mouse neutrophils toward the chemokine CXCL‐1 through TNF‐α‐stimulated VE‐cadherin WT (WT) or VE‐cadherin Y731F (Y731F) primary endothelial cells, each transfected with control or SHP2‐specific siRNA. The transmigration rate is presented relative to that of WT cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α‐tubulin. Transmigration of human neutrophils toward the chemokine IL‐8 through TNF‐α‐stimulated human endothelial cells (HUVEC), transfected with control or SHP2‐specific siRNA, and were either not transduced (−) or transduced (+) with adenoviruses expressing human SHP2 (Adv‐SHP2). The transmigration rate is presented relative to that of cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α‐tubulin. Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST, GST‐SHP2‐WT, or GST‐SHP2‐DA. Pulldowns and total cell lysates were analyzed by Western blotting for VE‐cadherin and GST. Graph below represents quantification of two independent experiments, presented as fold change of bound VE‐cadherin. TNF‐α‐activated bEnd.5 cells were incubated with mouse T cells for 0–30 min. SHP2 (above) or VE‐cadherin (below) were immunoprecipitated (IP) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. Graph below represents quantification of five independent experiments, presented as fold change of bound VE‐cadherin. Data information: Immunoblots are representative of two (C) or five (D) independent experiments and transmigration experiments represent data from four (A) and three (B) independent experiments with 3–6 values for each group per assay. Graphs represent mean ± SEM. Statistical significance was tested with one‐way ANOVA, (A, B) or Kruskal–Wallis test (D), * P < 0.05, ** P < 0.01, **** P < 0.0001, n.s., not significant. Source data are available online for this figure.

Article Snippet: VE‐cadherin was either incubated with phosphatase buffer only or with phosphatase buffer containing 2 µg recombinant human SHP2 (Sigma, SRP0217) for 30 min at 300 rpm and 30°C in a thermo‐shaker.

Techniques: Transmigration Assay, Transfection, Western Blot, Expressing, Transduction, Incubation, Immunoprecipitation, Molecular Weight

Journal: The EMBO Journal

Article Title: PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

doi: 10.15252/embj.2020106113

Figure Lengend Snippet: A Transmigration of mouse neutrophils toward CXCL‐1 through TNF‐α‐stimulated bEnd.5 cells transfected with control or SHP2‐specific siRNA and pre‐treated with anti‐endomucin (7C7.1) or anti‐PECAM‐1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%. B Transmigration of mouse neutrophils toward CXCL‐1 through TNF‐α‐stimulated WT or Y731F primary mouse endothelial cells pre‐treated with isotype control or anti‐PECAM‐1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%. C–E Extravasated leukocytes (C), adherent leukocytes (D), and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE‐cadherin mice stimulated intrascrotally with IL‐1β and treated i.v. with isotype control or anti‐PECAM‐1 (2H8) antibodies for 4 h before intravital microscopy. Data information: Graphs represent data from three (A, B) independent experiments in triplicate for each group (mean ± SEM). Data in (C–E) are of 38 vessels from four mice, 39 vessels from four mice, 31 vessels from three mice and 43 vessels from four mice analyzed in WT + IgG, WT + 2H8, Y731F + IgG, and Y731F + 2H8 treatment groups, respectively (mean ± SEM). Statistical significance was tested with one‐way ANOVA, * P < 0.05, ** P < 0.01, **** P < 0.0001, n.s., not significant.

Article Snippet: VE‐cadherin was either incubated with phosphatase buffer only or with phosphatase buffer containing 2 µg recombinant human SHP2 (Sigma, SRP0217) for 30 min at 300 rpm and 30°C in a thermo‐shaker.

Techniques: Transmigration Assay, Transfection, Intravital Microscopy

Journal: The EMBO Journal

Article Title: PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

doi: 10.15252/embj.2020106113

Figure Lengend Snippet: HUVEC were transduced with increasing titers of adenovirus expressing human SHP2. VE‐cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. bEnd.3 cells were lysed, and VE‐cadherin or immature VE‐cadherin was immunoprecipitated, respectively, with C5 or VD47 antibodies. Precipitates were blotted against pY731‐VE‐cadherin, β‐catenin, plakoglobin, p120, α‐catenin, and VE‐cadherin. VE‐cadherin–catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731‐VE‐cadherin, plakoglobin, β‐catenin, and VE‐cadherin. Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE‐cadherin set as 1. Molecular weight markers are indicated in kDa. Data information: Immunoblots are representative of at least three (A, B‐right panel, C) or five (B‐left panel) independent experiments (mean ± SEM). Statistical significance was tested with unpaired two‐tailed t ‐test, * P < 0.05, n.s., not significant. Source data are available online for this figure.

Article Snippet: VE‐cadherin was either incubated with phosphatase buffer only or with phosphatase buffer containing 2 µg recombinant human SHP2 (Sigma, SRP0217) for 30 min at 300 rpm and 30°C in a thermo‐shaker.

Techniques: Transduction, Expressing, Immunoprecipitation, Western Blot, Recombinant, Molecular Weight, Two Tailed Test

Journal: The EMBO Journal

Article Title: PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

doi: 10.15252/embj.2020106113

Figure Lengend Snippet: Transmigration of mouse neutrophils toward the chemokine CXCL‐1 through TNF‐α‐stimulated WT or Y731F primary endothelial cells, either pre‐treated with vehicle control (DMSO) or Ca 2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO‐treated endothelial cells expressing WT VE‐cadherin. TNF‐α‐activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen‐stimulated mouse T cells for 30 min. VE‐cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments is shown below, with the signal intensity for the control sample (no T cells, vehicle control) set as 100%. Transmigration assays as in (A), except for replacing MAPTAM treatment with Blebbistatin. Similar as for (B), except for replacing MAPTAM treatment with Blebbistatin. HUVEC transduced with lacZ or with SHP2 were either untreated (−) or treated (+) with thrombin for 30 min followed by immunoblotting either VE‐cadherin immunoprecipitates or cell lysates for the indicated antigens on the right. A quantification of four independent experiments is shown on the right. Molecular weight markers are indicated in kDa. Data information: The immunoblots are representative of five (B) or four (D, E) independent experiments, and the quantifications of transmigration experiments represent data from three (A, C) independent experiments in triplicate for each group per assay. Bars and error bars indicate mean ± SEM. Statistical significance was tested with one‐way ANOVA (B, E) or 2‐way ANOVA (A, C), *** P < 0.001, **** P < 0.0001, n.s., not significant. Source data are available online for this figure.

Article Snippet: VE‐cadherin was either incubated with phosphatase buffer only or with phosphatase buffer containing 2 µg recombinant human SHP2 (Sigma, SRP0217) for 30 min at 300 rpm and 30°C in a thermo‐shaker.

Techniques: Transmigration Assay, Expressing, Incubation, Immunoprecipitation, Western Blot, Transduction, Molecular Weight

Journal: The EMBO Journal

Article Title: PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

doi: 10.15252/embj.2020106113

Figure Lengend Snippet: Quantification of FRET efficiency (percentage) at junctions of HUVEC treated with siRNAs for SHP2 and VE‐cadherin prior to transducing VE‐cadherin‐FL. Cells were exposed either to flow alone or to flow in the presence of PMNs and then fixed with 4% PFA followed by FLIM measurements at sites of PMN transmigration (PMN) or at junctions without PMNs (Flow). Representative immunoblot for SHP2 and actin expression of HUVEC used for the experiments in (A). HUVEC were grown on collagen‐coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa (left) and 20 kPa (right) and stimulated with TNF‐α for 17 h prior to adding HL60‐derived neutrophils for 20 min. PECAM‐1 immunoprecipitates and cell lysates were immunoblotted for the indicated antigens. Note that leukocytes triggered SHP2 dissociation from PECAM‐1 independent of substrate stiffness. Data information: Graph in (A) represents n = 21 and 20 measurements in a total of four independent experiments. Bars and error bars indicate mean ± SEM. Statistical significance was tested with unpaired t ‐test, ** P < 0.01. Blots are representative for 3 experiments (C).

Article Snippet: VE‐cadherin was either incubated with phosphatase buffer only or with phosphatase buffer containing 2 µg recombinant human SHP2 (Sigma, SRP0217) for 30 min at 300 rpm and 30°C in a thermo‐shaker.

Techniques: Transmigration Assay, Western Blot, Expressing, Derivative Assay

Journal: The EMBO Journal

Article Title: PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

doi: 10.15252/embj.2020106113

Figure Lengend Snippet: Leukocytes destabilize endothelial junctions by stimulating PECAM‐1. Leukocyte‐induced stimulation of PECAM‐1 triggers the release of SHP2 that directly interacts with and dephosphorylates VE‐cadherin‐Y731, which is required for the internalization of VE‐cadherin. In addition to mobilizing SHP2, leukocytes stimulate Ca 2+ ‐signals in endothelial cells, and activate actomyosin‐based tension across the VE‐cadherin–catenin complex which is needed for Y731 dephosphorylation to occur. Since β‐catenin/plakoglobin mask the accessibility of Y731 for SHP2, we propose that leukocytes destabilize junctions by PECAM‐1‐SHP2‐triggered dephosphorylation of VE‐cadherin‐Y731 which becomes accessible by actomyosin‐mediated mechanical force exerted on the VE‐cadherin–catenin complex.

Article Snippet: VE‐cadherin was either incubated with phosphatase buffer only or with phosphatase buffer containing 2 µg recombinant human SHP2 (Sigma, SRP0217) for 30 min at 300 rpm and 30°C in a thermo‐shaker.

Techniques: De-Phosphorylation Assay

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Decreased expression of SHP2 in SSc. a The mRNA levels of SHP2 are significantly reduced in SSc skin as compared to healthy skin ( n = 7). b Immunohistochemistry of SHP2 in SSc skin and matched healthy controls. Representative images are shown at 200- and 1000-fold magnification. c Immunofluorescence staining of SHP2 with co-staining for the fibroblast marker P4Hβ, the endothelial cell marker CD31 and the leukocyte marker CD45, and DAPI. SSc fibroblasts demonstrated a reduced staining for SHP2 compared to healthy control. Representative images are shown at 400-fold magnification. Immunofluorescence pictures were processed to generate Voronoi tessellated pictures amenable to computational simulation. Quantification of SHP2 staining intensity ( n = 5) and of SHP2-positive cells ( n = 5). d , e The mRNA ( n = 5) ( d ) and protein level ( n = 4) ( e ) of SHP2 are decreased in cultured SSc fibroblasts. Horizontal scale bar, for all images, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. SSc: systemic sclerosis, Healthy: healthy individual, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Marker, Control, Cell Culture, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: SHP2 is downregulated in TGFβ signaling. a , b Decreased mRNA ( n = 6) ( a ) and protein ( n = 4) ( b ) levels of SHP2 in healthy fibroblasts stimulated with TGFβ (10 ng/ml) for different time points as measured by RT-PCR and western blot, respectively. c , d Overexpression of TBRI CA (6.67 × 10 7 IFUs every 2 weeks) significantly reduced mRNA ( n = 8) and the protein levels of Shp2 in murine skin as shown by qPCR ( c ) and immunofluorescence staining ( d ) of Shp2 with co-staining for fibroblast marker Vimentin and DAPI ( n ≥ 6 per each group). Representative images are shown at 100–200- and 600-fold magnification. Horizontal scale bar, 500 μm. Immunofluorescence pictures were analyzed by Voronoi tessellation. e , f Treatment with the selective TGFβ receptor type 1 kinase inhibitor SD208 (60 mg/kg/day) reversed the decrease of Shp2 mRNA ( n = 6) ( e ) and protein ( n = 4) ( f ) in bleomycin-challenged mice (50 µg every other day). g , h Treatment with the selective TGFβ receptor type 1 kinase inhibitor SD-208 reversed the decrease of Shp2 mRNA ( n = 6) ( g ) and protein ( h ) in TSK1 mice (2 mg tamoxifen over 5 days) ( n ≥ 6 per each group). i Phosphatase activity assay. Increases in SHP2 activity after TGFβ stimulation (10 ng/ml) ( n = 4) in cultured fibroblasts and upon overexpression of TGFβRI (6.67 × 10 7 IFUs) in murine skin ( n ≥ 4 per each group). Results shown are representative of three independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. AdLacZ: adenovirus LacZ, TBRI CA : constitutively active TGFβ receptor type I, TSK1: Tight skin, Bleo: bleomycin, Pa/Pa: control for TSK1, fluo.: fluorescence, int.: intensity, Unst.: unstimulated

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Immunofluorescence, Staining, Marker, Phosphatase Assay, Activity Assay, Cell Culture, MANN-WHITNEY, Control, Fluorescence

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Shp2 regulates TGFβ induced fibroblast activation. a Western blot for efficiency of Cre-mediated (80 IFUs/cell) knockout of Shp2 in murine Shp2 fl/fl fibroblasts ( n ≥ 3 per each group). b Shp2 knockout decreased mRNA levels of Acta2 ( n = 6). c – e Shp2 knockout decreased α-SMA and stress fiber staining. Representative images are shown at 200-fold magnification ( c ). Horizontal scale bar, 500 μm. Quantification of α-SMA staining intensity ( d ) and stress fiber staining intensity ( e ) ( n ≥ 3 different lines). f , g Col1a1 mRNA ( f ) and collagen protein release ( g ) induced by TGFβ (10 ng/ml for 24 h) ( n ≥ 3 different lines). Results shown are representative of three independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01* or # ; 0.01 > p > 0.001**; p < 0.001*** or ### ; ns: not significant. Significance was determined by Mann–Whitney test. AdCre: adenovirus Cre, AdLacZ: adenovirus LacZ, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Activation Assay, Western Blot, Knock-Out, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Fibroblast-specific knockout of Shp2 protects from experimental fibrosis. a TBRI CA -induced fibrosis (6.67 × 10 7 IFUs every 2 weeks). Representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. All groups consisted of ≥9 mice each. b Bleomycin-induced skin (50 µg every other day) fibrosis. Representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. All groups consisted of ≥8 mice each. c TSK1 model (2 mg tamoxifen over 5 days). Representative images of Masson trichrome-stained skin shown at 40-fold magnification. Hypodermal thickness, hydroxyproline content and myofibroblast counts. All groups consisted of ≥8 mice each. Horizontal scale bar in all images, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001** or ## ; p < 0.001***; ns: not significant. AdLacZ: adenovirus LacZ, TBRI CA : constitutively active TGFβ receptor type I, TSK1: Tight skin, Bleo: bleomycin, Shp2 Ko: SHP2 fibroblast-specific knockout

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Knock-Out, Staining

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Knockout of Shp2 decreases JAK2/STAT3 signaling. a Knockout of Shp2 in fibroblasts ( Shp2 fl/fl x Col1a2 ; Cre ER) decreases the levels of pJAK2 and pSTAT3 and STAT3 reporter activity in cultured fibroblasts ( n = 3 different lines). Cells were stimulated with TGFβ (10 ng/ml for 6 h). b – d Conditional knockout of Shp2 reduces the levels of pJAK2 and pSTAT3 in TBRI CA (6.67 × 10 7 IFUs every 2 weeks) ( b ) and bleomycin-induced fibrosis (50 µg every other day) ( c ) and in TSK1 mice (2 mg tamoxifen over 5 days) ( d ) ( n ≥ 3). Results shown are representative of three independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01* or # ; 0.01 > p > 0.001** or ## ; p < 0.001***; ns: not significant. Significance was determined by Mann–Whitney test. AdLacZ or LacZ: adenovirus LacZ, TBRI CA or TBRI: constitutively active TGFβ receptor type I, TSK1: Tight skin, Bleo: bleomycin, Tam: tamoxifen, Co: Control unstimulated, AdCre: adenovirus Cre

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Knock-Out, Activity Assay, Cell Culture, MANN-WHITNEY, Control

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: SHP2 enhances TGFβ-induced fibroblast activation via JAK2/STAT3. a mRNA levels of SHP2 after overexpression in human dermal fibroblasts. mRNA levels of COL1A1 in human fibroblasts transfected with empty vector, SHP2 WT - and SHP2 C459S -expression vectors, with or without TGFβ1 treatment (10 ng/ml for 24 h) ( n ≥ 4). b Western blot analysis and respective quantifications for type I collagen and SHP2 in human fibroblasts transfected with empty vector, SHP2 WT - and SHP2 C459S -expression vectors, with or without TGFβ1 treatment (10 ng/ml for 24 h). Western blot for pJAK2 Y1007/Y1008 , pJAK2 Y570 , total JAK2, pSTAT3 Y705 and total STAT3 with β-actin as loading control (TGFβ 10 ng/ml for 6 h) ( n = 3). Results shown are representative of three independent experiments. c , d Representative images of immunofluorescence stainings for α-SMA and stress fiber staining are shown at 400-fold magnification ( c ) and quantification of α-SMA staining intensity as well as stress fiber staining intensity ( d ) ( n ≥ 4). Horizontal scale bar, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. Vector: empty vector, SHP2 WT : plasmid carrying full length of SHP2 wild-type gene, SHP2 C459S : plasmid carrying a phosphatase-dead mutant of SHP2 , unstim.: unstimulated, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Activation Assay, Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Immunofluorescence, Staining, MANN-WHITNEY, Mutagenesis

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Overexpression of JAK2 ∆Y570F prevents the inhibitory effects of SHP2 inhibitors on TGFβ-induced fibroblast activation. a mRNA levels of COL1A1 and COL1A2 (TGFβ 10 ng/ml for 24 h) ( n ≥ 5). b Release of collagen protein (TGFβ 10 ng/ml for 24 h) ( n ≥ 6). c Representative images of immunofluorescence stainings for α-SMA and stress fiber at 400-fold magnification and respective quantifications (TGFβ 10 ng/ml for 24 h) ( n ≥ 6). Horizontal scale bar, 500 μm. d STAT3 reporter Assay upon JAK2 WT and Y570F mutant overexpression. Cells were treated with TGFβ (10 ng/ml for 6 h) and NSC-87877 (100 µM) ( n ≥ 4). e Co-immunoprecipitation and respective quantifications of endogenous JAK2 with endogenous SHP2 in human fibroblasts stimulated with TGFβ (10 ng/ml for 30′) ( n = 3). Results shown are representative of ≥ 3 independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***; ns: not significant. Significance was determined by Mann–Whitney test. JAK2 WT : JAK2 Wild type, JAK2 ΔY570F : JAK2 mutant resistant to phosphorylation at Y570, NSC-87877: SHP1/SHP2 inhibitor, Co: control unstimulated, int.: intensity, IP: immunprecipitation

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Over Expression, Activation Assay, Immunofluorescence, Reporter Assay, Mutagenesis, Immunoprecipitation, MANN-WHITNEY, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Inhibition of SHP2 limits JAK2/STAT3 signaling and fibroblast activation. a Changes in the mRNA levels of COL1A1 and of collagen protein in human fibroblasts incubated with increasing doses of NSC-87877 (10 µM, 30 µM and 100 µM). Fibroblasts were treated with TGFβ (10 ng/ml) for 24 h. b ACTA2 mRNA. ( n ≥ 4) c Representative images of immunofluorescence stainings for α-SMA and stress fiber staining are shown at 400-fold magnification and quantification of α-SMA staining intensity as well as stress fiber staining intensity ( n ≥ 15) (TGFβ 10 ng/ml for 24 h). Horizontal scale bar, 500 μm. d Representative western blots for pJAK2 Y1007/Y1008 , pJAK2 Y570 , total JAK2, pSTAT3 Y705 and total STAT3 with β-actin as loading control and quantification of the results (TGFβ 10 ng/ml for 6 h) ( n ≥ 2). e Changes in STAT3 reporter activity ( n ≥ 6) (TGFβ 10 ng/ml for 6 h). Results shown are representative of three independent experiments All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***; ns: not significant. Significance was determined by Mann–Whitney test. NSC-87877: SHP1/SHP2 inhibitor, Co: control unstimulated, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Inhibition, Activation Assay, Incubation, Immunofluorescence, Staining, Western Blot, Control, Activity Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Treatment with NSC-87877 ameliorates experimental fibrosis. The SHP1/SHP2 inhibitor NSC-87877 was applied at doses of 5 mg/kg q.d. a Bleomycin-induced skin (50 µg every other day) fibrosis: representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. b TBRI CA -induced (6.67 × 10 7 IFUs every 2 weeks) skin fibrosis: representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. c Bleomycin-induced lung fibrosis (50 µg single doses): representative images of Sirius red-stained lung shown at 100-fold magnification. Quantification of Sirius red-positive area (fibrotic area), hydroxyproline content and myofibroblast counts. All groups in all models consisted of ≥5 mice each. Horizontal scale bar in all images, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test; Bleo: bleomycin, TBRI CA : constitutively active TGFβ receptor type I, AdLacZ: adenovirus LacZ

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Selective inhibition of Shp2 ameliorates experimental fibrosis. The following doses of SHP2 inhibitors were applied: PHPS1 (5 mg/kg q.d.), SHP099 (75 mg/kg q.d.) and 11-a1 (7.5 mg/kg q.d.). a Bleomycin-induced pulmonary fibrosis (50 µg single doses) : representative images of Masson trichrome-stained skin shown at 100-fold magnification; fibrotic area, hydroxyproline content and myofibroblast counts. b TBRI CA -induced dermal fibrosis (6.67 × 10 7 IFUs every 2 weeks): representative images of Masson trichrome-stained skin shown at 100-fold magnification; Dermal thickness, myofibroblast counts and hydroxyproline content. All groups in both models consisted of ≥4 mice each. Horizontal scale bar for all images, 500 μm All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. Bleo: bleomycin, AdLacZ: adenovirus LacZ, TBRI CA : constitutively active TGFβ receptor type I

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Inhibition, Staining, MANN-WHITNEY