recombinant protein  (R&D Systems)

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: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: SHP-2 is activated in response to force on E-cadherin. (A–G) MCF10A cells, or MCF10A cells expressing shRNAs against SHP-2 (shSHP-2) or a scramble shRNA sequence (scSHP-2) were incubated with paramagnetic beads coated with IgG or E-cadherin extracellular domains (Ecad). Cells were left resting (no force, NF) or tensile force was applied (+), and the cells were lysed immediately (1 min) or at the indicated times (Min. after Force). (A–C) SHP-2 is activated by force, and loss of SHP-2, E-cadherin or mechanical signaling prevents activation. Lysates were immunoblotted for SHP-2 Y542 phosphorylation (pSHP-2) or total SHP-2. In C, the cells were pre-incubated with a myosin II inhibitor [blebbistatin (Blebbi)] or E-cadherin function-blocking antibody (HECD-1) prior to application of force. (D,E) Vinculin is phosphorylated when SHP-2 is inactive and dephosphorylated when SHP-2 is active. Lysates from the cells were immunoblotted with phospho-specific antibodies that recognize Y822 vinculin (pY822) or total vinculin. (F,G) SHP-2 is recruited to the cadherin adhesion complex in response to force. The magnetic beads were recovered, and co-precipitating levels of pSHP-2 were examined. In G, the cells were pre-treated with a myosin II inhibitor (Blebbi) or an E-cadherin function-blocking antibody (HECD-1) prior to application of force. (H,I) Shear stress elicits similar responses in SHP-2 activation and vinculin Y822 phosphorylation as tensile force. Shear stress was applied to cells, the cells were lysed at the indicated times, and the lysates were immunoblotted for pSHP-2 or total SHP-2 (H), or pY822 or total vinculin (I). The graphs beneath the immunoblots in all panels represent the quantification of a minimum of three independent experiments±s.e.m. *P<0.05.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Expressing, shRNA, Sequencing, Incubation, Activation Assay, Blocking Assay, Magnetic Beads, Shear, Western Blot

Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: SHP-2 specifically targets phospho-Y822 vinculin. (A) Vinculin binds a SHP-2 trapping mutant. MCF10A cells were left resting (−) or pre-treated with pervanadate and then lysed. The lysates were incubated with a substrate-trapping mutant, GST-SHP-2 (D425A/C459A). The co-precipitating levels of vinculin were monitored by immunoblotting, and the levels of the trapping mutant were visualized by Coomassie Blue staining of the gel. (B,C) Vinculin binding to the SHP-2 trapping mutant is blocked by mutation of Y822F. GFP fusions of wild-type (WT), Y100F, Y822F or Y1065F vinculin were expressed in MCF10A cells, and the levels of each expressed protein were examined by immunoblotting with tubulin as a loading control in B. In C, lysates from the indicated cell lines were incubated with the SHP-2 trapping mutant and the products of the reactions were examined as described in A. (D) Force increases vinculin trapping by SHP-2. Cells were left resting (no force, NF) or tensile force was applied and the cells were lysed immediately (1 min) or 10 min later. Substrate trapping was measured as described in A. (E) SHP-2 dephosphorylates Y822 vinculin in vitro. Cells were left untreated or treated with pervanadate. Vinculin was immunoprecipitated, and the immunoprecipitates were subjected to an in vitro phosphatase assay in the presence (+) or absence (−) of recombinant SHP-2. Levels of vinculin phosphorylated at Y822 (pY822) relative to total immunoprecipitated vinculin were monitored by immunoblotting. Graphs adjacent to each image represent the quantification of a minimum of three independent experiments±s.e.m. *P<0.05.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Mutagenesis, Incubation, Western Blot, Staining, Binding Assay, In Vitro, Immunoprecipitation, Phosphatase Assay, Recombinant

Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: Inhibition of SHP-2 cells elevates mechanotransduction. (A) A schematic of the E-cadherin-mediated cell-stiffening cascade. Force on E-cadherin activates a mechanosignaling pathway, whereby Abl phosphorylates vinculin at Y822. Vinculin Y822 phosphorylation is essential for E-cadherin-dependent RhoA-mediated contractility, which culminates in phosphorylation of MLC and heightened actomyosin contractility. (B) SHP-2 expression is inhibited in cells expressing shRNAs. Two mass populations of cells expressing shRNAs against SHP-2 (shSHP-2 and shSHP-2.2) were generated and examined by immunoblotting for SHP-2 levels relative to a loading control (p34-Arc subunit of the Arp2/3 complex). The graphs below each image represent the quantification of a minimum of three independent experiments±s.e.m. (C,D) Mechanotransduction is elevated in cells with depressed SHP-2 levels. Lysates from parental or shSHP-2 cells were immunoblotted for vinculin phosphorylated at Y822 (pY822) or total vinculin in C, and MLC phosphorylated at T18 and S19 (pMLC) or total MLC in D. The graphs below each image represent the quantification of a minimum of three independent experiments±s.e.m. (E–G) SHP-2 inhibition increases actin reinforcement. MCF10A parental or shSHP-2 cells were left resting or subjected to shear stress as described in the Materials and Methods. The cells were fixed and stained with antibodies against β-catenin, or with Texas-Red Phalloidin, and examined by confocal microscopy (E). Graphs beside images represent the average corrected fluorescence intensity of F-actin (G) or β-catenin (F) in the cell–cell junctions. Fifty junctions were quantified for each condition. Box-and-whisker plots represent the 10th, 25th, 50th, 75th and 90th percentiles. *P<0.05. Scale bar: 10 µm.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Inhibition, Expressing, Generated, Western Blot, Shear, Staining, Confocal Microscopy, Fluorescence, Whisker Assay

Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: Mutation of the residues N-terminal of Y822 creates a vinculin mutant that is constitutively phosphorylated. (A) A schematic depicting the amino acid sequences surrounding vinculin Y822. SHP-2 prefers substrates with acidic residues at the −3 and −2 positions. In vinculin, the −2 and −3 positions are occupied by the acidic residues, S and D. (B) The mutant vinculins were expressed at similar levels in MCF10A cells. Full-length vinculin harboring the indicated mutations was stably expressed as a fusion with GFP in MCF10A cells. The levels of expression were examined by immunoblotting for GFP and tubulin as a loading control. (C) The mutant vinculins do not bind SHP-2. The cells expressing the mutant vinculins were pre-treated with pervanadate for 30 min. Lysates were incubated with GST-SHP-2 (D425A/C459A) and the co-precipitating levels of GFP-vinculin were monitored by immunoblotting. (D) Mutant vinculins retain phosphorylation at Y822. The mutant vinculins were immunoprecipitated. Levels of vinculin Y822 phosphorylation were monitored by immunoblotting with antibodies against phospho-Y822, and then the blots were stripped and re-probed for total vinculin levels. (E) Mutant vinculins are recruited to the E-cadherin adhesion complex. The mutant vinculins were immunoprecipitated. Levels of co-precipitating E-cadherin were monitored by immunoblotting. The graphs in each panel represent the quantification of at least three independent experiments±s.e.m. *P<0.05.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Mutagenesis, Stable Transfection, Expressing, Western Blot, Incubation, Immunoprecipitation