shp2 activity assay kit  (BPS Bioscience)

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