Journal: PLoS ONE

Article Title: Insulin-Stimulated Degradation of Apolipoprotein B100: Roles of Class II Phosphatidylinositol-3-Kinase and Autophagy

doi: 10.1371/journal.pone.0057590

Figure Lengend Snippet: Primary hepatocytes from Apobec1 −/− mice were cultured in serum free conditions for 16 h before the addition of insulin to a final concentration of 100 nM. At the indicated times, the cells were harvested in ice cold 0.5 M TCA, and acidic lipids were extracted. PI(3,4)P 2 concentrations were measured by dot blotting using anti-PI(3,4)P 2 antibody (n = 3 at each time point). Data represent the mean±SEM.

Article Snippet: Cellular PI(3,4)P 2 concentrations were measured by using anti-PI(3,4)P 2 mouse monoclonal antibody (Echelon Biosciences) following manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay

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: PLoS ONE

Article Title: Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis

doi: 10.1371/journal.pone.0055393

Figure Lengend Snippet: Standard plot of absorbance at 450 nm versus (A) log pmol PI(4,5)P2 and (C) log pmol PI3P. B Percentage change in PI(4,5)P2 content after BL irradiation in control (10 mM PIPES), neomycin (250 µM) and U73122 (25 µM) samples. D Percentage change in PI3P level after BL irradiation in control (10 mM PIPES), WM (10 µM) and LY294002 (200 µM). The vertical bars represent SE of 3 independent experiments.

Article Snippet: In a manner similar to PI(4,5)P2, the effects of WM and LY294002 on PI3K were also tested using the Echelon Bioscience mass Elisa kit (K3300).

Techniques: Irradiation

Journal: PLoS ONE

Article Title: Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis

doi: 10.1371/journal.pone.0055393

Figure Lengend Snippet: Phot1 induction by weak BL activates PI3K and PI4K, thereby producing PI3P and PI4P respectively. PI4P can be hydrolyzed by PLC generating water soluble Ins(1,4)P2. On the other hand, phot2 triggers activation of PI3K, PI4K and the PI(4,5)P2-PLC pathway upon weak BL induction, and only the PI(4,5)P2-PLC pathway upon strong BL. IPPs can be stepwise phosphorylated by inositolpolyphosphate multikinases, IPKs, to produce inositol hexaphosphate (InsP6), which has been also shown to be linked with Ca 2+ mobilization . One of the modes of action for PI3P, PI4P and PI(4,5)P2 during chloroplast relocations is calcium release in the cytoplasm. ↑ represents Ca 2+ transient rise. PIP5K: phosphatidylinositol-4-phosphate 5-kinase and IPK: inositol polyphosphate kinase.

Article Snippet: In a manner similar to PI(4,5)P2, the effects of WM and LY294002 on PI3K were also tested using the Echelon Bioscience mass Elisa kit (K3300).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

doi: 10.1371/journal.pone.0033889

Figure Lengend Snippet: ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

Techniques: Injection, Expressing, Dominant Negative Mutation

Journal: PLoS ONE

Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

doi: 10.1371/journal.pone.0033889

Figure Lengend Snippet: Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

Techniques: