phosphoinositol  (Echelon Biosciences)

Journal: The Journal of Biological Chemistry

Article Title: Structural Basis of Phosphoinositide Binding to Kindlin-2 Protein Pleckstrin Homology Domain in Regulating Integrin Activation *

doi: 10.1074/jbc.M111.295352

Figure Lengend Snippet: Structure Determination of the Kindlin-2 PH Domain Bound to IP4

Article Snippet: Uniformly 15 N-labeled or 15 N/13 C-labeled K2-PH protein (1 m m ) was dissolved in 10 m m Hepes, pH 6.8, 50 m m NaCl, 2 m m DTT with or without 2 m m IP4 (Echelon Bioscience Inc., Salt Lake City, UT, catalogue number Q-1345) in 90% H2 O/10% (v/v) D2 O or 99.96% D2 O. Inositol 1,4,5-triphosphate (IP3, catalogue number Q-0145) was also used for chemical shift mapping analysis.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Structural Basis of Phosphoinositide Binding to Kindlin-2 Protein Pleckstrin Homology Domain in Regulating Integrin Activation *

doi: 10.1074/jbc.M111.295352

Figure Lengend Snippet: Structure-based sequence alignment of K2-PH domain with other selective PH domain structures bound to IP4. Residues involved in binding to different phosphate groups are colored differently: yellow , binding to the 1-phosphate group; red , binding to the

Article Snippet: Uniformly 15 N-labeled or 15 N/13 C-labeled K2-PH protein (1 m m ) was dissolved in 10 m m Hepes, pH 6.8, 50 m m NaCl, 2 m m DTT with or without 2 m m IP4 (Echelon Bioscience Inc., Salt Lake City, UT, catalogue number Q-1345) in 90% H2 O/10% (v/v) D2 O or 99.96% D2 O. Inositol 1,4,5-triphosphate (IP3, catalogue number Q-0145) was also used for chemical shift mapping analysis.

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural Basis of Phosphoinositide Binding to Kindlin-2 Protein Pleckstrin Homology Domain in Regulating Integrin Activation *

doi: 10.1074/jbc.M111.295352

Figure Lengend Snippet: Solution structure of the complex between the kindlin-2 PH domain and IP4. Left , ensemble of 20 lowest energy structures of the kindlin-2 PH domain bound to IP4. Right , ribbon drawing of a representative structure of the kindlin-2 PH domain bound to IP4

Article Snippet: Uniformly 15 N-labeled or 15 N/13 C-labeled K2-PH protein (1 m m ) was dissolved in 10 m m Hepes, pH 6.8, 50 m m NaCl, 2 m m DTT with or without 2 m m IP4 (Echelon Bioscience Inc., Salt Lake City, UT, catalogue number Q-1345) in 90% H2 O/10% (v/v) D2 O or 99.96% D2 O. Inositol 1,4,5-triphosphate (IP3, catalogue number Q-0145) was also used for chemical shift mapping analysis.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Structural Basis of Phosphoinositide Binding to Kindlin-2 Protein Pleckstrin Homology Domain in Regulating Integrin Activation *

doi: 10.1074/jbc.M111.295352

Figure Lengend Snippet: Chemical shift perturbation profiles of wild type and mutant K2-PH with IP4. Gray bars indicate line-broadening residues. The perturbation profiles were derived from the HSQC spectra of 0.1 m m 1 H- 15 N-labeled wild type and mutant samples in the absence

Article Snippet: Uniformly 15 N-labeled or 15 N/13 C-labeled K2-PH protein (1 m m ) was dissolved in 10 m m Hepes, pH 6.8, 50 m m NaCl, 2 m m DTT with or without 2 m m IP4 (Echelon Bioscience Inc., Salt Lake City, UT, catalogue number Q-1345) in 90% H2 O/10% (v/v) D2 O or 99.96% D2 O. Inositol 1,4,5-triphosphate (IP3, catalogue number Q-0145) was also used for chemical shift mapping analysis.

Techniques: Mutagenesis, Derivative Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Structural Basis of Phosphoinositide Binding to Kindlin-2 Protein Pleckstrin Homology Domain in Regulating Integrin Activation *

doi: 10.1074/jbc.M111.295352

Figure Lengend Snippet: K2-PH domain has a positively charged IP4 binding pocket. Residues that have potential contacts with IP4 are highlighted and labeled. The phosphate groups on IP4 are also labeled. The dotted line indicates how the phosphate group 1 can be extended to

Article Snippet: Uniformly 15 N-labeled or 15 N/13 C-labeled K2-PH protein (1 m m ) was dissolved in 10 m m Hepes, pH 6.8, 50 m m NaCl, 2 m m DTT with or without 2 m m IP4 (Echelon Bioscience Inc., Salt Lake City, UT, catalogue number Q-1345) in 90% H2 O/10% (v/v) D2 O or 99.96% D2 O. Inositol 1,4,5-triphosphate (IP3, catalogue number Q-0145) was also used for chemical shift mapping analysis.

Techniques: Binding Assay, Labeling

Journal: Infection and Immunity

Article Title: Phosphatidylinositol-(3,4,5)-Trisphosphate Induces Phagocytosis of Nonmotile Pseudomonas aeruginosa

doi: 10.1128/IAI.00215-18

Figure Lengend Snippet: PIP 3 treatment of cells and integration in the plasma membrane are essential for phagocytic induction of nonmotile P. aeruginosa . (A) Murine WT BMDCs were assayed for relative phagocytosis of PA14 motAB motCD (MOI of 10) without or after treatment of bacteria (gray bar) or cells (white bar) with 12 μM PIP 3 . The data were normalized to phagocytosis of PA14 motAB motCD without PIP 3 treatment and are represented as fold increase in phagocytosis. (B) Phagocytic uptake of P. aeruginosa PA14 motAB motCD (MOI of 10) by murine WT BMDCs in the absence or presence of 12 μM inositol 1,3,4,5-tetrakisphosphate (IP 4 ). Phagocytic uptake was normalized as a percentage of the mean phagocytosis of motAB motCD bacteria in untreated cells. (C) The killing rate of PA14 WT and motAB motCD bacteria by BMDCs treated with PIP 3 was assayed by a gentamicin protection assay. Following a 45-min incubation of bacteria with BMDCs (MOI of 10), gentamicin was added, and at the indicated time points following gentamicin addition (20, 40, and 60 min), cells were lysed to assess remaining CFU counts. The number of CFU recovered was plotted relative to recovery at 20 min after gentamicin addition. All data were analyzed using one-way ANOVA with Tukey's post hoc analyses (A) or using an unpaired t test with Welch's correction (B) and are representative of at least two independent biological experiments ( n ≥8). ***, P ≤ 0.0005; *, P ≤ 0.05; ns, not significant, for results compared to those without PIP 3 treatment.

Article Snippet: For studies using inositol 1,3,4,5-tetrakisphosphate (IP4 ) (Q-1345; Echelon Biosciences), 12 μM phosphoinositol was added to the cells at the same time as the bacteria.

Techniques: Incubation