Journal: PLoS ONE

Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

doi: 10.1371/journal.pone.0013396

Figure Lengend Snippet: HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, PtdIns(4,5) P 2 .

Article Snippet: Di-C8 phosphoinositides (Echelon Biosciences Inc. catalogue numbers P-3008, P-5008, and P-3508) were used as substrates and PTEN lipid phosphatase (Echelon Biosciences Inc. catalogue number E-3000) was used as a positive control.

Techniques: Isolation, In Vivo

Journal: PLoS ONE

Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

doi: 10.1371/journal.pone.0013396

Figure Lengend Snippet: Thin layer chromatographic separation of the products of the in vitro phosphorylation reaction between PIP4Kάandendogenous phosphoinositides isolated from leaves (lane 2), stems (lane 3), flowers (lane 4) and siliques (lane 5). A PtdIns(4,5) P 2 standard is shown (lane 1) (panel a). HPLC analysis of deacylated products of the in vitro phosphorylation reaction between endogenous phosphoinositides (from leaves) and PIP4Kα. Deacylated PtdIns(3,4) P 2 and PtdIns(4,5) P 2 used as standards (panel b). The products of the in vitro phosphorylation reaction between endogenous phosphoinositides isolated from rosette leaves and PIP4Kα were incubated in the absence (lane 1) or presence (lane 2) of yeast YNK-5 phosphatase. The reaction products were analyzed by thin layer chromatography. The position of a PtdIns4 P standard is shown (lane 3) (panel c).

Article Snippet: Di-C8 phosphoinositides (Echelon Biosciences Inc. catalogue numbers P-3008, P-5008, and P-3508) were used as substrates and PTEN lipid phosphatase (Echelon Biosciences Inc. catalogue number E-3000) was used as a positive control.

Techniques: In Vitro, Isolation, Incubation, Thin Layer Chromatography

Journal: PLoS ONE

Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

doi: 10.1371/journal.pone.0013396

Figure Lengend Snippet: Nuclear localisation of ePHD-GFP expressed alone. Arrows in all panels point to nuclei (panel a). ePHD-GFP co-expressed with wild-type AtMTM1-RFP causes depletion of the green nuclear signal (panel b). The consensus amino acids in the active AtMTM1 phosphatase site and the amino acid substitutions in mut1 and mut2 (panel c). Phosphoinositide 3′-phosphatase activity of recombinant wild type, mut1 and mut2 phosphatases. The Vmax values towards PtdIns(3,5) P 2 as a substrate are shown as the percentage of the wild-type (WT) ATMTM1 activity. Bars are s.d. from three measurements (panel d). Distribution of ePHD-GFP co-expressed with mut1AtMTM1-RFP show nuclear localizations of the green signal (panel e). Co-expression of ePHD-GFP with mut2AtMTM1-RFP. The green signal is largely distributed in the cytoplasm (panel f). Bars are 50 Pm.

Article Snippet: Di-C8 phosphoinositides (Echelon Biosciences Inc. catalogue numbers P-3008, P-5008, and P-3508) were used as substrates and PTEN lipid phosphatase (Echelon Biosciences Inc. catalogue number E-3000) was used as a positive control.

Techniques: Activity Assay, Recombinant, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Structural Basis of Membrane Targeting by the Dock180 Family of Rho Family Guanine Exchange Factors (Rho-GEFs)

doi: 10.1074/jbc.M110.102517

Figure Lengend Snippet: Phosphoinositide binding to the DHR-1 domain. A, the head group of PtdIns(3,4,5)P3 with phosphate positions numbered. B, thermodynamic parameters for phosphoinositide and head group binding to wild-type DHR-1 (Kd, dissociation constant; ΔH, enthalpy change; TΔS, temperature (K) × entropy change; ΔG (the free energy change) = ΔH-TΔS = −RT ln Kd); N is the apparent stoichiometry. No binding was observed for PtdIns(3,5)P2, PtdIns(3,4)P2, or PtdIns(3)P1. * indicates the experiment was performed in 145 mm NaCl. All other experiments were carried out in low salt (see “Experimental Procedures”). Representative ITC profiles are provided in supplemental Fig. S4. C, effect of point mutations on PtdIns(3,4,5)P3 binding, defined as Kd(wt)/Kd(mutant). Error estimates are from fitting of ITC titration curves. At right are melting temperatures, Tm, for each mutant, in °C. *, the Tm value given for K524A is actually for the triple mutant, K446A/K524A/K555A. D, mutation sites mapped onto the DHR-1 structure. E and F, ITC competition titrations (negative values of energy indicate an exothermic reaction; positive values, endothermic). E, DHR-1 equilibrated with a 1.5 m excess of PtdIns(4,5)P2 titrated with PtdIns(3,4,5)P3. F, the converse experiment: DHR-1/PtdIns(3,4,5)P3 titrated with PtdIns(4,5)P2.

Article Snippet: Each titration involved 19 injections of 2-μl aliquots of ∼1 m m water-soluble di-C8 phosphoinositide analogs (Echelon Biosciences, Salt Lake City, UT) or InsP 4 into cells containing 25–100 μ m DHR-1 at 23 °C.

Techniques: Binding Assay, Mutagenesis, Titration