Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Chemical structure of RMC-4550 and X-ray crystal structure of SHP2 in complex with RMC-4550 (PDB code 7RCT). Surface representation of SHP2 in complex with RMC-4550 bound in the central tunnel formed at the interface of N-SH2 (green), C-SH2 (blue) and PTP (wheat) domains. (B) Chemical structures of RMC-4550-based PROTAC candidates, R1–1C, R1–3C and R1–5C.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques:

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Inhibition of SHP2-F285S- or PTP-mediated DIFMUP dephosphorylation by R1–1C, R1–3C, R1–5C and RMC-4550. MV4;11 cells were treated with increasing doses of R1–3C (B), R1–1C or R1–5C (C) for 24 h and subjected to Western blotting using SHP2, GAPDH and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Time course of SHP2 degradation by R1–5C (100 nM) in MV4;11 cells. Immunoblotting with SHP2 and β-actin antibodies. (B) CRBN−/− and parental MOLT4 cells were treated with increasing doses of R1–5C for 24 h and subjected to Western blotting using SHP2, CRBN and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A-D) Scatterplots displaying relative fold-change in SHP2 abundance following treatment of MV4;11 cells with 100 nM R1–5C for 4 h (A), 8 h (B), 16 h (C) or 100 nM RMC-4550 (D). SHP2/PTPN11 is highlighted in red. Hits highlighted in blue in (C) and (D) indicate changes in abundance of proteins at 16 h time point due to secondary effects (such as transcriptional responses) of SHP2 degradation or inhibition. (E) Heatmap of the protein abundance changes in MV4;11 cells comparing treatment with 100 nM R1–1C (4 h and 16 h), 100 nM R1–3C (4 h and 16 h), 100 nM R1–5C (2 h, 4 h, 8 h and 16 h), 100 nM RMC-4550 (16 h) and 1 μM pomalidomide (5 h). The heatmap colors are scaled with red indicating a decrease in protein abundance (−2 log2 FC) and blue indicating an increase (2 log2 FC) in protein abundance.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: SHP2 is identified as a PLCγ-dependent VEGFR2 pY1175 interaction partner in endothelial cells (A) Representative western blots showing VEGFR2 immunoprecipitated with antibodies against RASA1, CSK, VAV2, or SHP2 in HUVECs unstimulated (−) or stimulated (+) with VEGFA (100 ng/mL, 5 min). Corresponding whole-cell lysates analyzed using antibodies against phosphorylated VEGFR2 (pY1175), total VEGFR2, and GAPDH as loading control. (B) Quantification of VEGFR2 interaction with RASA1, CSK, VAV2, and SHP2 from (A); n = 3 independent experiments. (C) Representative images of immunostainings for VEC (magenta), pVEC Y685 (green), and DAPI (blue) of unstimulated or VEGFA-stimulated (100 ng/mL, 5 min) HUVECs, pretreated with siCtr or siPTPN11. Scale bars: 30 μm. (D and E) Quantifications of MFI from (C), displayed as fold change relative to unstimulated control. (D) MFI of the VEC area. (E) MFI of pVEC Y685; n = 3 independent experiments, ≥3 fields of view per experiment. (F) Representative images of immunostainings for VEC (magenta), pVEC Y685 (green), and DAPI (blue) of unstimulated or VEGFA-stimulated (100 ng/mL, 5 min) HUVECs, pretreated with siCSK or siVAV2. Scale bars: 30 μm. (G and H) Quantifications of MFI from (F), shown as fold change over unstimulated control; n = 3 independent experiments, ≥3 fields of view per experiment. (I) Representative western blot showing VEGFR2 immunoprecipitated with antibodies against SHP2 or IgG control from unstimulated or VEGFA-stimulated (100 ng/mL, 5 min) HUVECs, pre-treated with siCtr or siPLCG1 . Corresponding whole-cell lysates analyzed by blotting with antibodies against pVEGFR2 Y1175, VEGFR2, PLCγ, and GAPDH as loading control. (J) Quantification of VEGFR2-SHP2 binding from (I); n = 3 independent experiments. (K) Representative western blot showing downstream VEGFA-induced signaling in unstimulated (−) or 100 ng/mL VEGFA-stimulated HUVECs, for 2, 5, 10, and 20 min pretreated with siCtr or siPTPN11. (L–N) Quantification of western blots from (K), shown as fold change relative to unstimulated control. (L) Quantification of western blots for peNOS S1177. (M) Quantification of western blots for pSFK Y418. (N) Quantification of western blots for pVEGFR2 Y1175; n = 4–5 independent experiments. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. HUVECs, human umbilical vein endothelial cells; VEC, VE-cadherin; MFI, mean fluorescence intensity. See also .

Article Snippet: The primary antibodies used for pull-down were all produced in mice and include anti-CSK (Invitrogen Thermo Fisher, MA5-15707), anti-PTPN11 (SHP2) (Origene, TA501914), anti-RASA1 (Thermo Fisher Scientific, MA4-001), anti-VAV2 (Invitrogen Thermo Fisher, MA5-38657), and IgG1 (k isotope) (BD Pharmingen, 555746).

Techniques: Western Blot, Immunoprecipitation, Control, Binding Assay, Fluorescence

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: Endothelial PLCγ/SHP2 signaling mediates activation of Src by regulation of both the activating and inhibitory tyrosine phosphorylation sites (A) PLA using antibodies against Src and pSFK Y418 to detect phosphorylation of Src on Y418 in unstimulated or VEGFA-stimulated HUVECs (100 ng/mL, 5min), pretreated with siCtr or siPTPN11 . Endothelial junctions are stained for VEC (magenta) and nuclei with DAPI (blue). Scale bars: 30 μm. Boxed regions in the upper panels are shown at higher magnification in panels below. Scale bars: 10 μm. (B and C) MFI quantifications from (A), displayed as fold change relative to unstimulated control. (B) MFI of the total PLA signal. (C) MFI of the junctional PLA signals representing Y418 phosphorylation of Src; n = 7 independent experiments, ≥3 fields of view per experiment. (D) Representative western blot showing pSFK Y529 signaling in unstimulated (−) or VEGFA-stimulated HUVECs (100 ng/mL) for 2, 5, 10 and 20 min, pretreated with siCtr or si PTPN11 . (E) Quantification of western blots from (D); n = 5 independent experiments. (F and G) PLA using antibodies against Src and pSFK 529, visualizing phosphorylation of Src at the inhibitory phosphosite in HUVECs stimulated for 2, 5, and 10 min or left unstimulated. (F) Phosphorylation of Src at the inhibitory phosphosite in HUVECs pre-treated with siCtr . (G) Phosphorylation of Src at the inhibitory phosphosite in HUVECs pre-treated with siPTPN11 . Endothelial junctions are stained for VEC (magenta) and nuclei with DAPI (blue). Scale bars: 30 μm. Boxed regions in the upper panels are shown at a higher magnification in panels below. Scale bars: 10 μm. (H) Quantification of PLA experiments from (F) and (G) and F; n = 6 independent experiments, ≥3 fields of view per experiment. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. PLA, proximity ligation assay; HUVECs, human umbilical vein endothelial cells; VEC, VE-cadherin. See also .

Article Snippet: The primary antibodies used for pull-down were all produced in mice and include anti-CSK (Invitrogen Thermo Fisher, MA5-15707), anti-PTPN11 (SHP2) (Origene, TA501914), anti-RASA1 (Thermo Fisher Scientific, MA4-001), anti-VAV2 (Invitrogen Thermo Fisher, MA5-38657), and IgG1 (k isotope) (BD Pharmingen, 555746).

Techniques: Activation Assay, Phospho-proteomics, Staining, Control, Western Blot, Proximity Ligation Assay

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: PLCγ/SHP2 interplay leads to eNOS activation followed by Src nitration (A) Representative western blot showing eNOS T495 signaling in unstimulated (−) or 100 ng/mL VEGFA-stimulated HUVECs for 2, 5, 10 and 20 min, pre-treated with siCtr or siPTPN11 . (B) Quantification of western blots from (A); n = 4 independent experiments. (C) PLA for NitroTyr and pSFK Y418 to detect full activation of Src in HUVECs stimulated with VEGFA (100 ng/mL, 5 min) or left unstimulated, and pretreated with siCtr or siPTPN11 . Endothelial junctions are stained for VEC (magenta) and DAPI (blue). Scale bars: 30 μm. Boxed regions in the upper panels are shown at higher magnification in panels below. Scale bars: 10 μm. (D) Quantification of junctional MFI PLA signals representing Y418 phosphorylation and 3-nitration of Src from (C), displayed as fold change to unstimulated control; n = 5 independent experiments, ≥3 fields of view per experiment. (E) Western blot showing eNOS S1177 and SHP2 Y542 signaling in unstimulated (−) or 100 ng/mL VEGFA-stimulated HUVECs for 2, 5, 10, and 20 min, pre-treated with siCtr or siPLCG1 . (F and G) Quantifications of western blots from (E). (F) Quantifications of western blots for peNOS S1177. (G) Quantifications of western blots for pSHP2 Y542; n = 4 independent experiments. (H) Representative immunostaining images with antibodies against VE-cadherin (VEC; magenta) and pSHP2 Y542 (green), in HUVECs unstimulated or stimulated with VEGFA (100 ng/mL, 5min) after downregulation with siCtr or siPLCG1 . Scale bars: 30 μm. (I) Quantification of MFI from (H), shown as fold change relative to unstimulated control; n = 4 independent experiments, ≥3 fields of view/experiment. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. VEC, VE-cadherin. See also .

Article Snippet: The primary antibodies used for pull-down were all produced in mice and include anti-CSK (Invitrogen Thermo Fisher, MA5-15707), anti-PTPN11 (SHP2) (Origene, TA501914), anti-RASA1 (Thermo Fisher Scientific, MA4-001), anti-VAV2 (Invitrogen Thermo Fisher, MA5-38657), and IgG1 (k isotope) (BD Pharmingen, 555746).

Techniques: Activation Assay, Nitration, Western Blot, Staining, Phospho-proteomics, Control, Immunostaining

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: VEGFR2 pY1173/PLCγ-induced vascular permeability in vivo requires SHP2 (A) Representative immunostaining images with antibodies against VEC (magenta) and pSHP2 Y542 (green) in the back skin of WT and Plcg1 iECKO mice after intradermal injection of PBS or VEGFA. Scale bars: 50 μm. (B) Quantification of MFI values from (A), for vascular pSHP2 Y542, displayed as fold change relative to PBS control; n = 5 mice/genotype, ≥3 fields of view/mouse. (C) Representative images of immunostaining for VEC (magenta) and pVEC Y685 (green) in the back skin of WT mice, intradermally injected with DMSO or SHP099 and subsequently PBS or VEGFA at the same site. Scale bars: 50 μm. (D) Quantification of MFI values from (C), for vascular pVEC Y685, shown as fold change relative to PBS control; n = 5 (DMSO) and 4 (SHP099) WT mice, ≥3 fields of view/mouse. (E) The Miles assay showing Evans blue leakage in the back skin of DMSO (control) or SHP099 treated WT mice, intradermally injected with PBS or VEGFA. (F) Quantification of extravasated Evans blue from (E), shown as fold change of DMSO-PBS-treated mice; n ≥ 8 mice/condition. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. VEC, VE-cadherin. See also .

Article Snippet: The primary antibodies used for pull-down were all produced in mice and include anti-CSK (Invitrogen Thermo Fisher, MA5-15707), anti-PTPN11 (SHP2) (Origene, TA501914), anti-RASA1 (Thermo Fisher Scientific, MA4-001), anti-VAV2 (Invitrogen Thermo Fisher, MA5-38657), and IgG1 (k isotope) (BD Pharmingen, 555746).

Techniques: Permeability, In Vivo, Immunostaining, Injection, Control

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: VEGFR2 Y1173 heterozygosity is accompanied by decreased tumor endothelial PLCγ/SHP2 signaling and tumor vascular leakage (A) Representative immunostaining images of Vegfr2 +/+ (WT) and Vegfr2 Y1173F/+ (Y1173F/+) B16F10 melanoma tumors, showing vessels (IB4; red), pSHP2 Y542 (yellow), and fibrinogen (green). (B and C) Quantification of MFI from (A), shown as fold change over WT. (B) MFI for pSHP2 Y542. (C) MFI for fibrinogen; n = 5 (WT) and 3 (Y1173F/+) mice, ≥3 fields of view/mouse. (D) Representative immunostaining images of Vegfr2 +/+ (WT) and Vegfr2 Y1173F/+ (Y1173F/+) B16F10 melanoma tumors, showing vessels (CD31; red), pVEC Y731 (green), and CD45 (cyan). (E and F) Quantification of MFI from (D), displayed as fold change of WT. (E) MFI for pVEC Y731. (F) MFI for CD45 + cells (F); n = 5 (WT) and 3 (Y1173F/+), ≥3 fields of view/mouse. Scale bars: 100 μm. (G) Representative immunostaining images of WT and Plcg1 iECKO B16F10 melanoma tumors, showing vessels (IB4; red), pSHP2 Y542 (yellow), and fibrinogen (green). (H and I) MFI quantifications from (G), shown as fold change of WT. (H) MFI for pSHP2 Y542. (I) MFI for fibrinogen; n = 7 (WT) and 7 ( Plcg1 iECKO ), ≥3 fields of view/mouse. Unpaired 2-tailed Student’s t test. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. MFI, mean fluorescence intensity. See also .

Article Snippet: The primary antibodies used for pull-down were all produced in mice and include anti-CSK (Invitrogen Thermo Fisher, MA5-15707), anti-PTPN11 (SHP2) (Origene, TA501914), anti-RASA1 (Thermo Fisher Scientific, MA4-001), anti-VAV2 (Invitrogen Thermo Fisher, MA5-38657), and IgG1 (k isotope) (BD Pharmingen, 555746).

Techniques: Immunostaining, Fluorescence

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: SHP2 and PLCγ are clinically relevant biomarkers for tumor vascular leakage (A and B) Representative multiplex images of RCC patient biopsies. (A) Multiplex images for RCC patients with low vascular PLCγ and SHP2 expression. (B) Multiplex images for RCC patients with high vascular PLCγ and SHP2 expression. Tissue sections were stained for PLCγ (red), SHP2 (yellow), CD34 (green), and FpA (white) to visualize vascular leakage, and counterstained with Hoesht. Scale bars: 100 μm. Boxed regions are shown at higher magnification to the right. Scale bars: 30 μm. (C) Heatmap comparing endothelial SHP2 expression, PLCγ expression, and vascular leakage scores across individual RCC patients ( n = 16). Patients are arranged by their ID number, with color coding indicating high (green), medium (yellow), or low (red) levels. (D) Schematic for the mechanism of SHP2/PLCγ interaction upon VEGFR2 activation. In summary, VEGFA stimuli leads to the phosphorylation of VEGFR2 Y1175, which recruits both PLCγ and SHP2. SHP2 is needed for the activation of PLCγ at the plasma membrane and hydrolysis of PIP2 to IP3 and DAG. The former triggers the release of intracellular calcium (Ca 2+ ), and together with DAG, activates PKC, causing phosphorylation of the eNOS activating site S1177 and dephosphorylation of the inactivating site T495. Production of NO mediates nitration and full activation of Src, which phosphorylates Y685 of VEC, disrupting the adherens junctions and resulting in vascular leakage. Created in BioRender. Kremmyda, P. (2025). RCC, renal cell carcinoma; FpA, fibrinopeptide A; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; VEC, VE-cadherin. See also .

Article Snippet: The primary antibodies used for pull-down were all produced in mice and include anti-CSK (Invitrogen Thermo Fisher, MA5-15707), anti-PTPN11 (SHP2) (Origene, TA501914), anti-RASA1 (Thermo Fisher Scientific, MA4-001), anti-VAV2 (Invitrogen Thermo Fisher, MA5-38657), and IgG1 (k isotope) (BD Pharmingen, 555746).

Techniques: Multiplex Assay, Expressing, Staining, Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, De-Phosphorylation Assay, Nitration

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r 2 = 0.987). ( B ) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). ( C ) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( D ) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. ( E ) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys 333 and Cys 367 . ( F ) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. ( G ) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. .

Article Snippet: Protein-containing samples in SDS-PAGE sample buffer were subjected to electrophoresis and immunoblotting using the following primary antibodies: SFN (1:1000, in-house), Shp2 (1:1000, Abcam #32083) for recombinant protein or (R&D Systems #AF1894) for immunoprecipitation experiments, GAPDH (1:5000, CST #2118) and an anti-rabbit secondary antibody (1:2500, CST #7074).

Techniques: Recombinant, Activity Assay, Incubation, Western Blot, Modification, Residue, Immunoprecipitation, Mutagenesis, Transfection