vitro phosphatase assays active shp2  (SignalChem)

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

Figure Lengend Snippet: IRS1/2 are required for insulin-activated IR endocytosis. a HepG2 cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP antibodies. b Quantification of the ratios of PM and IC IR-GFP signals of cells in a (siLUC, n = 26; siLUC + Ins, n = 42; si-IRS1, n = 41; si-IRS1, n = 26; si-IRS2, n = 20; si-IRS2 + Ins, n = 10; si-IRS1/2, n = 48, si-IRS1/2 + Ins, n = 66; si-IRS1/2 + IRS1, n = 14; si-IRS1/2 + IRS1 + Ins, n = 14; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). c Domains and YXXΦ motifs of human IRS1 and mouse IRS2. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. AP2M1- and SHP2-binding regions are indicated. YXXΦ motifs and phosphotyrosine sites of IR for SHP2 binding are shown as blue and red bars, respectively. The MAPK phosphorylation sites are labeled as green letters in the sequences. d 293FT cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP (IR; green), anti-Myc (IRS1; red), and DAPI (blue) (3YA, the IRS1 Y612A/Y632A/Y662A triple mutant; 3YF, the Y612F/Y632F/Y662F triple mutant; 3SA, the S616A/S636A/S666A triple mutant; 3SD, the S616D/S636D/S666D triple mutant; 2YA, the Y1179A/Y1229A double mutant). Scale bar, 5 µm. e Quantification of the ratios of PM and IC IR-GFP signals of cells in d (siLUC, n = 49; siLUC + Ins, n = 87; si-IRS1/2, n = 46; si-IRS1/2 + Ins, n = 87; si-IRS1/2 + WT, n = 65; si-IRS1/2 + WT + Ins, n = 112; si-IRS1/2 + 3YA, n = 54; si-IRS1/2 + 3YA + Ins, n = 63, si-IRS1/2 + 3SA, n = 40; si-IRS1/2 + 3SA + Ins, n = 66; si-IRS1/2 + 3 SD, n = 42; si-IRS1/2 + 3 SD + Ins, n = 63; si-IRS1/2 + 3YF, n = 48; si-IRS1/2 + 3YF + Ins, n = 58; si-IRS1/2 + Y612A, n = 48; si-IRS1/2 + Y612A, n = 58; si-IRS1/2 + 1–864, n = 23; si-IRS1/2 + 1–864 + Ins, n = 35; si-IRS1/2 + 2YA, n = 28, si-IRS1/2 + 2YA + Ins, n = 42; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). f 293FT cells were serum starved and treated without or with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP, and IgG IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. g Serum-starved primary hepatocytes were treated with DMSO or 10 µM SHP099 for 2 h and treated with 100 nM insulin for 5 min. TCL, anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Two Tailed Test, Binding Assay, Phospho-proteomics, Labeling, Mutagenesis

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

Figure Lengend Snippet: The SHP2-MAPK pathway promotes insulin-activated IR endocytosis. a HepG2 cells expressing IR-GFP WT were starved, treated with the indicated inhibitors for 2 h, treated without or with 100 nM insulin for 20 min, and stained with anti-GFP (IR; green) and DAPI (blue). Scale bar, 5 µm. b Quantification of the ratios of PM and IC IR-GFP signals of cells in a (DMSO, n = 47; DMSO + Ins, n = 56; IRi, n = 50; IRi + Ins, n = 65; U0126, n = 96; U0126 + Ins, n = 111; PD0325901, n = 26; PD0325901 + Ins, n = 54; SHP099, n = 43; SHP099 + Ins, n = 49; GDC0941, n = 57; GC0941 + Ins, n = 48; AKTi, n = 65; AKTi + Ins, n = 96; BI2536, n = 26; BI2536 + Ins, n = 24; Reversine, n = 37; Reversine + Ins, n = 27; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). c Binding of IRS1 peptides to AP2M1 (residues 160–435). Input and proteins bound to IRS1 peptide beads were analyzed by SDS-PAGE and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± s.d.; n = 5 independent experiments). d Isothermal titration calorimetry (ITC) analysis of binding between IRS1 peptides and AP2M1 (residue 160–435), with K d indicated. e The IRS1 peptides were incubated with active SHP2 for the indicated durations, spotted onto membranes, and detected with the anti-pY612-IRS1 antibody. f Quantification of the relative SHP2 activity in e (mean ± s.d.; n = 4 independent experiments; **** p < 0.0001; two-tailed unpaired t test). g Model of the regulation of insulin-activated IR endocytosis by a phosphorylation switch on IRS1/2. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and MAPK pathways. SHP2 acts upstream of RAS-RAF and promotes the activation of MAPK pathway. p31 comet binds to the IR-bound MAD2 and blocks IR-AP2 association to prevent premature IR endocytosis. In feedback regulation, activated ERK1/2 phosphorylate S616 and other sites on IRS1. SHP2 binds to the C-terminal phosphotyrosine site on IRS1 and dephosphorylates pY612 of the doubly phosphorylated IRS1 (pY612/pS616), thus promoting IRS1–AP2M1 association. p31 comet is released from MAD2 by an unknown mechanism, allowing the assembly of an MCC-like complex on IR. MAD2- and IRS1/2-dependent AP2 recruitment and clustering trigger clathrin-mediated IR endocytosis. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Expressing, Staining, Two Tailed Test, Binding Assay, SDS Page, Isothermal Titration Calorimetry, Residue, Incubation, Activity Assay, Phospho-proteomics, Activation Assay

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

Figure Lengend Snippet: SHP2 inhibition delays IR endocytosis and improves insulin sensitivity in mice. a , b Glucose tolerance test ( a ) and insulin tolerance test ( b ) of male mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. At 1 day after the last drug administration, experiments were performed. For GTT ( a ), vehicle, n = 12; SHP099, n = 10; for ITT ( b ), vehicle, n = 11; SHP099, n = 10; mean ± s.e.m; two-tailed unpaired t test. c Body weight of mice administered vehicle or SHP099 at 7 days post administration. Mean ± s.d. d HFD-fed mice were administered vehicle or SHP099 for 5 days. The mice were fasted overnight and again administered vehicle or SHP099. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 μm. e Quantification of the ratios of plasma membrane (PM) and intracellular compartments (IC) IR signals of the livers in d (vehicle, 0 min, n = 43; 1 min, n = 54; 5 min, n = 27; 15 min, n = 36 and SHP099, 0 min, n = 35; 1 min, n = 43; 5 min, n = 30; 10 min, n = 33; 15 min, n = 33; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). f – h The levels of fasting serum insulin ( f ) and C-peptide ( g ), and the ratio of C-peptide:insulin ( h ) in mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. Vehicle, n = 11; SHP099, n = 12. i HepG2 cells stably expressing IR-GFP were transfected with CEACAM1 siRNAs, serum starved, treated without or with 100 nM insulin form 5 min, and stained with anti-GFP and DAPI. Quantification of the ratios of PM and IC IR-GFP signals of cells was shown (siLUC, n = 28; siLUC + Ins, n = 49; siCEACAM1 #1 + Ins, n = 27; siCEACAM1 #2 + Ins, n = 25; siCEACAM1 #3 + Ins, n = 35; siCEACAM1 #4 + Ins, n = 18; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). j Western blot analysis of cell lysates in i . k Model of the regulation of insulin-activated IR endocytosis. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Inhibition, Two Tailed Test, Injection, Staining, Clinical Proteomics, Membrane, Stable Transfection, Expressing, Transfection, Western Blot

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

Figure Lengend Snippet: Mitotic regulators and SHP2 promote feedback inhibition of IR. a Insulin signaling in the liver from mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 5 days, fasted overnight, and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b Quantification of the blots in a . Mean ± range; n = 2 independent experiments. c Targeting feedback regulation of IR endocytosis for diabetes treatment. Left panel depicts the feedback regulation of IR endocytosis by ERK1/2 and SHP2 during unperturbed insulin signaling. Right panel illustrates the mechanism by which SHP2 inhibitor (SHP099) or shRNA blocks growth-promoting IR signaling and IR endocytosis, and prolongs insulin signaling through the PI3K-AKT pathway, which controls metabolism. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Inhibition, Injection, Western Blot, shRNA

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

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The Toxoplasma secreted effector Tg WIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2

doi: 10.1007/s00018-024-05283-3

Figure Lengend Snippet: Phosphorylated Tg WIP binds to host cell tyrosine phosphatases Shp1 and Shp2. A Schematic showing the amino acid sequence of Tg WIP. Magenta color indicates the position of the three tyrosines in Tg WIP. Orange region represents the WRC interacting receptor sequence (WIRS) domain that could mediate Tg WIP’s interaction with the WAVE complex. Blue regions represent the proline rich regions (PRRs) that could mediate Tg WIP’s interactions with Nck and Grb2. B DCs were infected with parasites expressing wild-type Tg WIP ( Tg WIP WT ), or Tg WIP in which its SH2-binding motifs Y150 ( Tg WIP Y150A ), Y199 ( Tg WIP Y199A ), or both ( Tg WIP Y150A/Y199A ) are mutated to an alanine. Shown are the Western blots using phosphorylated tyrosine (pY) (1st membrane-probing antibody), HA (2nd membrane-probing antibody after stripping membrane), and Shp2 (3rd membrane-probing antibody after stripping membrane twice) antibodies on total lysate or immunoprecipitated Tg WIP. C The murine DC (DC2.4) and human foreskin fibroblast (HFF) cell lines were infected with type II ME49 Δtgwip complemented with HA-tagged wild-type Tg WIP ( Tg WIP WT ) for 4 h (intracellular) or until parasite egress (extracellular). Lysates were generated and Tg WIP immunoprecipitated. Shown are the Western blots using phosphorylated tyrosine (pY), HA, Shp1, and Shp2 antibodies, (probed in the same order as in C). D DC2.4 were treated with the Src kinase inhibitor Dasatinib at the indicated concentrations for 1 h and throughout infection with WT Toxoplasma . DMSO is the solvent control. Western blot analysis using antibodies against pY and HA. DCs were infected with Tg WIP WT or with either E Tg WIP Y150A or with F Tg WIP Y150A/Y199A Toxoplasma. Shown are the Western blots using antibodies against Shp2 in E and Shp1 in F . G-H In vitro phosphatase assay of Shp2 in G Shp1 in H incubated with recombinant Tg WIP, with or without prior phosphorylation by recombinant Src kinase. Reaction rates, represented by linear fluorescence changes over time, were normalized against the rate of Shp2 or Shp1 without Tg WIP. Data from 3 biological repeats are indicated by different colors, with each data point showing the average from three technical repeats. Bar graph represents the mean and standard error of the 3 biological repeats. ANOVA with Tukey’s post hoc test was used. n = 3; *** for p < 0.0005 against the first column). n.s.: not significant. I-J Coomassie blue-stained SDS-PAGE gel showing GST-tagged, Src-phosphorylated Tg WIP (GST-TgWIP P ) pulling down MBP-tagged full-length (FL) or individual SH2 domains of Shp2 in I or Shp1 in J . Loading controls contain 6 pmol of indicated prey proteins

Article Snippet: The impact of phosphorylated Tg WIP on both Shp1 and Shp2 activity was measured using a Shp2 activity assay kit (BPS bioscience, Cat# 79,330 [ ]), following the manufacturer’s instructions.

Techniques: Sequencing, Infection, Expressing, Binding Assay, Western Blot, Membrane, Stripping Membranes, Immunoprecipitation, Generated, Solvent, Control, In Vitro, Phosphatase Assay, Incubation, Recombinant, Fluorescence, Staining, SDS Page

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1/2 are required for insulin-activated IR endocytosis. a HepG2 cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP antibodies. b Quantification of the ratios of PM and IC IR-GFP signals of cells in ( a ) (mean ± SD; *p<0.0001). c Domains and YXXΦ motifs of human IRS1 and mouse IRS2. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. AP2M1- and SHP2-binding regions are indicated. YXXΦ motifs and phosphotyrosine sites of IR for SHP2 binding are shown as blue and red bars, respectively. The MAPK phosphorylation sites are labeled as green letters in the sequences. d 293FT cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP (IR; green), anti-Myc (IRS1; red), and DAPI (blue). (3YA, Y612A/Y632A/Y662A; 3YF, Y612F/Y632F/Y662F; 3SA, S616A/S636A/S666A; 3SD, S616D/S636D/S666D; Y2A, Y1179A/Y1229A). e Quantification of the ratios of PM and IC IR-GFP signals of cells in ( d ) (mean ± SD; *p<0.0001). f 293FT cells were serum starved and treated without or with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP, and IgG IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. g Serum-starved primary hepatocytes were treated with DMSO or 10 µM SHP099 for 2 h and treated with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1 promotes IR endocytosis and interacts with AP2. a Western blot analysis of cell lysates in . Asterisks indicate non-specific bands. b Domains and YXXΦ motifs of human IRS1. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. IRS1 fragments that can or cannot bind to AP2M1 are presented as red or black lines, respectively. YXXΦ motifs and phosphotyrosine sites for SHP2 binding are presented as blue and red bars, respectively. c Binding of IRS1 WT and mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with anti-Myc (IRS1) antibodies and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=3 independent experiments). d Binding of IRS1 WT and truncation mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with the indicated antibodies. The relative band intensities are shown below (n=2 independent experiments). e Sequence alignment of a conserved region in IRS1/2. Three YXXΦ motifs are boxed with red dashed lines. The phosphorylation sites of IR and MAPK are indicated as red and blue dots, respectively.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Western Blot, Binding Assay, Staining, Sequencing

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: The SHP2-MAPK pathway promotes insulin-activated IR endocytosis. a HepG2 cells expressing IR-GFP WT were starved, treated with the indicated inhibitors for 2 h, treated without or with 100 nM insulin for 20 min, and stained with anti-GFP (IR; green) and DAPI (blue). b Quantification of the ratios of PM and IC IR-GFP signals of cells in (A) (mean ± SD; *p<0.0001). c Binding of IRS1 peptides to AP2M1 (residues 160-435). Input and proteins bound to IRS1-peptide beads were analyzed by SDS-PAGE and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=4 independent experiments). d Isothermal titration calorimetry (ITC) analysis of binding between IRS1 peptides and AP2M1 (residue 160-435), with K d indicated. e The IRS1 peptides were incubated with active SHP2 for the indicated durations, spotted onto membranes, and detected with the anti-pY612-IRS1 antibody. f Quantification of the relative SHP2 activity in ( e ) (mean ± SD; n=4 independent experiments; *p<0.0001). g Model of the regulation of insulin-activated IR endocytosis by a phosphorylation switch on IRS1/2. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and MAPK pathways. SHP2 acts upstream of RAS-RAF and promotes the activation of MAPK pathway. p31 comet binds to the IR-bound MAD2 and blocks IR-AP2 association to prevent premature IR endocytosis. In feedback regulation, activated ERK1/2 phosphorylate S616 and other sites on IRS1. SHP2 binds to the C-terminal phospho-tyrosine site on IRS1 and dephosphorylates pY612 of the doubly phosphorylated IRS1 (pY612/pS616), thus promoting IRS1-AP2M1 association. p31 comet is released from MAD2 by an unknown mechanism, allowing the assembly of an MCC-like complex on IR. MAD2- and IRS1/2-dependent AP2 recruitment and clustering trigger clathrin-mediated IR endocytosis. h Ribbon diagram of the crystal structure of AP2M1 (residues 160-435) bound to pS-IRS1. pS-IRS1 is shown as sticks. i Surface drawing of AP2M1, with pS-IRS1 shown as sticks. j A close-up view of the surface drawing of AP2M1 colored by its electrostatic potential (blue, positive; red, negative; white, neutral). pS-IRS1 is shown as sticks.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Expressing, Staining, Binding Assay, SDS Page, Isothermal Titration Calorimetry, Incubation, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: SHP2 inhibition delays IR endocytosis and improves insulin sensitivity in mice. a , b Glucose tolerance test ( a ) and insulin tolerance test ( b ) of male mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. At 1 day after the last drug administration, experiments were performed. Vehicle, n=12; SHP099, n=10; mean ± SEM. c Body weight of mice administered vehicle or SHP099 at 7 days post administration. Mean ± SD. d HFD-fed mice were administered vehicle or SHP099 for 5 days. The mice were fasted overnight and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. e Quantification of the ratios of plasma membrane (PM) and intracellular compartments (IC) IR signals of the livers in ( d ) (mean ± SD; *p<0.0001). f - h The levels of fasting serum insulin ( f ) and C-peptide ( g ), and the ratio of C-peptide:insulin ( h ) in mice fed normal chow or HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. i HepG2 cells stably expressing IR-GFP were transfected with CEACAM1 siRNAs, serum starved, treated without or with 100 nM insulin form 5 min, and stained with anti-GFP and DAPI. Quantification of the ratios of PM and IC IR-GFP signals of cells was shown (mean ± SD). j Western blot analysis of cell lysates in ( i ). k Model of the regulation of insulin-activated IR endocytosis by CEACAM1, the MAD2–CDC20– BUBR1 module, and the SHP2-IRS1/2 module.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Staining, Stable Transfection, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Depletion of SHP2 by shRNA delays IR endocytosis and improves insulin sensitivity in mice. a The level of SHP2 in liver, skeletal muscle and epididymal WAT from mice fed HFD for 5 weeks. The mice were injected with AAV-control (Ctrl) or SHP2 shRNA. At 17 days after injection, the mice were fasted overnight and injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. WAT and skeletal muscle were collected at 2 min and 3 min after the indicated time points, respectively. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b , c Glucose tolerance test ( b ) and insulin tolerance test ( c ) in mice injected with AAV-Ctrl or AAV-SHP2 shRNA and fed HFD. Experiments were performed at 2 weeks after injection. n=6; mean ± SEM. d Body weight in HFD-fed mice injected with AAV-Ctrl or AAV-SHP2 shRNA. Mean ± SD. e HFD-fed mice were injected with AAV-Ctrl or AAV-SHP2. At 17 days after injection, the mice were fasted overnight and injected with or without 1U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. f Quantification of the ratios of PM and IC IR signals of the livers in ( e ) (mean ± SD; *p<0.0001).

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: shRNA, Injection, Western Blot, Staining

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Mitotic regulators and SHP2 promote feedback inhibition of IR. a Insulin signaling in the liver from mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 5 days, fasted overnight, and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b Quantification of the blots in ( a ). Mean ± SD; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. c Targeting feedback regulation of IR endocytosis for diabetes treatment. Left panel depicts the feedback regulation of IR endocytosis by ERK1/2 and SHP2 during unperturbed insulin signaling. Right panel illustrates the mechanism by which SHP2 inhibitor (SHP099) or shRNA blocks growth-promoting IR signaling and IR endocytosis, and prolongs insulin signaling through the PI3K-AKT pathway, which controls metabolism.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Western Blot, shRNA