Figure 3
From: The TIPE Molecular Pilot That Directs Lymphocyte Migration in Health and Inflammation
TNFAIP8 binding to phosphoinositides, and its effect on PtdIns(3,4,5)P3 generation by PI3Ks. (a,b) SPR analysis of TNFAIP8 binding to DOPC membranes containing 10% (mole/mole) PtdIns(4,5)P2 (a) or PtdIns(3,4,5)P3 (b) on L1 sensor chip. Purified PLCδ-PH and GRP1-PH domains were used as positive controls, and trypsin inhibitor as a negative control (Control). The percent of maximal binding (Left Panels) and equilibrium KD (Right Panels) are shown. (c) Sedimentation-based phosphoinositide binding assay showing the proportion of TNFAIP8 and control protein trypsin inhibitor bound to SUVs containing 100% PC, 10% PtdIns(4,5)P2 or 10% PtdIns(3,4,5)P3. (d) Phosphorylation of PtdIns(4,5)P2 by PI3Ks as measured in the ADP-Glo kinase assay, in the presence of increasing concentrations of murine TNFAIP8 protein or 2 μM BSA (Control). The values are mean ± s.d. (a–c) or mean ± s.e.m. (d). Data represent three independent experiments (a,b) or are pooled from four (c) or two independent experiments done in duplicates (d). ***P < 0.001 (Student’s t-test (c)).
