Journal: Methods in Molecular Biology

Article Title: Cyclic Nucleotide Signaling in Plants

doi: 10.1007/978-1-62703-441-8

Figure Lengend Snippet: Fig. 2 Assay of CD38 activity with NGD as substrate. Upper panel : Continuous assay. 100 μ M NGD is incubated with CD38, 1 μ g/ml, in 20 mM Tris–HCl, pH 8, and the fl uorescence measured at excitation of 300 nm and emission of 410 nm. Lower panel : Discontinuous assay. A sample, such as a suspension of red blood cells (40 %) is incubated with 100 μ M NGD in HBSS. At the times indicated, the reaction is stopped by adding 0.6 M perchloric acid. The protein is removed by centrifugation at 10,000 × g for 10 min. The supernatant is recovered and the acid removed by extraction with 4 volumes of chloroform/tri- n -octylamine as described in the text. The neutral aqueous phase is recovered and fl uorescence measured at 300 nm excitation and 410 nm emission. The fl uorescence signal is quanti fi ed by comparing to cGDPR standards

Article Snippet: We use an inverted epi fl uorescence microscope (Diaphot-TMD; Nikon, http://www.nikon.com ) with a 40× air objective and a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan).

Techniques: Activity Assay, Incubation, Suspension, Centrifugation, Extraction

Journal: Methods in Molecular Biology

Article Title: Cyclic Nucleotide Signaling in Plants

doi: 10.1007/978-1-62703-441-8

Figure Lengend Snippet: Fig. 3 Measurement of NADase activity by fl uorescence assay with etheno-NAD. The NADase is prepared from N. crassa extracts as described in the text. 100 μ M etheno-NAD is added to 20 μ l of extract in 200 μ l of 25 mM Tris–HCl, pH 8, and 2 mM MgCl 2 and fl uorescence measured at 300 nm excitation and 410 nm emission

Article Snippet: We use an inverted epi fl uorescence microscope (Diaphot-TMD; Nikon, http://www.nikon.com ) with a 40× air objective and a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan).

Techniques: Activity Assay

Journal: Methods in Molecular Biology

Article Title: Cyclic Nucleotide Signaling in Plants

doi: 10.1007/978-1-62703-441-8

Figure Lengend Snippet: Fig. 5 Cycling assay for cADPR. ( a ) Time-dependent increases in fl uorescence for cADPR standards at 544 nm excitation and 590 nm emission. The numbers to the right refer to nM concentration of cADPR. ( b ) Standard curve constructed from the slopes of increase in fl uorescence versus cADPR concentration. ( c ) Time- dependent increase in fl uorescence for a sample prepared from red blood cells

Article Snippet: We use an inverted epi fl uorescence microscope (Diaphot-TMD; Nikon, http://www.nikon.com ) with a 40× air objective and a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan).

Techniques: Concentration Assay, Construct

Journal: Methods in Molecular Biology

Article Title: Cyclic Nucleotide Signaling in Plants

doi: 10.1007/978-1-62703-441-8

Figure Lengend Snippet: Fig. 2 Example of FlincG fl uorescence detection. Fluorescence was measured in one mesophyll protoplast and standardized by dividing the FlincG signal by its initial fl uorescence. After a short equilibration time, the fl uorescence became stable and addition of Jasmonic acid (1 nM, arrow ) to the protoplast buffer tran- siently induced a ~10 % increase of FlincG fl uorescence

Article Snippet: We use an inverted epi fl uorescence microscope (Diaphot-TMD; Nikon, http://www.nikon.com ) with a 40× air objective and a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan).

Techniques: Fluorescence

Journal: Methods in Molecular Biology

Article Title: Cyclic Nucleotide Signaling in Plants

doi: 10.1007/978-1-62703-441-8

Figure Lengend Snippet: Fig. 1 Fluorescence characteristics of the cameleon Ca 2+ reporter. Ca 2+ binds to the calmodulin (CaM) domain between the CFP (cyan fl uorescent protein) and YFP (yellow fl uorescent protein) causing it to bind to the M13 peptide. The resul- tant conformational change causes CFP to come into close proximity to YFP, allowing FRET ( fl uorescence resonance energy transfer) to occur and YFP fl uorescence to be produced from CFP excitation. As Ca 2+ levels increase, the 485-nm emission from CFP (FRET donor) decreases, whereas the 535-nm emis- sion from YFP (FRET acceptor) increases. The ratio of the 535-nm/485-nm (440-nm excitation) can be calibrated to give values to [Ca 2+ ] cyt.

Article Snippet: We use an inverted epi fl uorescence microscope (Diaphot-TMD; Nikon, http://www.nikon.com ) with a 40× air objective and a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan).

Techniques: Fluorescence, Förster Resonance Energy Transfer, Produced

Journal: Methods in Molecular Biology

Article Title: Cyclic Nucleotide Signaling in Plants

doi: 10.1007/978-1-62703-441-8

Figure Lengend Snippet: Fig. 1 Typical results obtained for NO detection using DAF-FM DA in maize roots [( a )–( e ), see ref. 8 ] and soybean root nodules [( g )–( l ), see ref. 9 ) ]. ( a ), ( d ), ( h ), and ( k ) are bright- fi eld images; ( b ), ( e ), ( g ), and ( j ) are fl uorescence images, whereas ( c ), ( f ), ( i ), and ( l ) are overlay images of the bright- fi eld and fl uorescence images. ( d ), ( e ), and ( f ) represent the control in which no DAF-FM DA was added (DAF-FM DA loading buffer lacking DAF-FM DA), whereas ( j ), ( k ), and ( l ) represent controls where 1 mM L-NNA was added before addition of the DAF-FM DA loading buffer

Article Snippet: We use an inverted epi fl uorescence microscope (Diaphot-TMD; Nikon, http://www.nikon.com ) with a 40× air objective and a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan).

Techniques: Control

Journal: Journal of Lipid Research

Article Title: Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

doi: 10.1194/jlr.m012591

Figure Lengend Snippet: Fig. 1. Phospholipase activity (A) and liposome binding (B) of wild-type (WT) and T178A mutant PlcHR 2 are shown. A: Wild-type or mutant PlcHR2 (0.5 g/ml) was assayed at 37°C on 0.3 mM LUV composed of PC:SM:PE:Chol (1:1:1:1, molar ratio). Phospho- lipase activity was measured as water-soluble lipid phosphorus. B: Binding of wild-type or mutant PlcHR2 was assayed as an increase in the enzyme’s intrinsic fl uorescence (F bound) in the presence of increasing concentrations of LUV of the composition indicated above. Data are average values ± SD (n = 3).

Article Snippet: After GUV formation, the chamber was placed on an inverted confocal fl uorescence microscope (D-Eclipse C1 model; Nikon Inc., Melville, NY).

Techniques: Activity Assay, Binding Assay, Mutagenesis

Journal: Journal of Lipid Research

Article Title: Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

doi: 10.1194/jlr.m012591

Figure Lengend Snippet: Fig. 3. Progressive PlcHR 2 binding to GUVs after initial binding detection. Experimental conditions are as described in the legend to Fig. 1 . Enzyme binding is measured as an increase in membrane- bound Alexa fl uorescence as a function of time. Time 0 is the mo- ment at which enzyme binding was fi rst detected. A: control; (B) with 10 mol% DAG; (C) with 10 mol% Cer; (D) with 5 mol% Cer plus 5 mol% DAG. Results are average values of 10 vesicles ± SEM.

Article Snippet: After GUV formation, the chamber was placed on an inverted confocal fl uorescence microscope (D-Eclipse C1 model; Nikon Inc., Melville, NY).

Techniques: Binding Assay, Membrane, Control