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  • Data experimental design materials and methods br

    2018-11-07

    Data, experimental design, materials and methods
    Experimental design Two Brassica napus genotype under two kinds of P treatments, long-term low P stress and short-term P-free starvation, were conducted, and three time points were used in each P treatment. Total protein was extracted from roots or leaves respectively of two B. napus by triplicate. 2-DE images were generated and compared to gain spots with abundance altered at least ±2-fold (T-test p<0.05). Then protein spots were identified by MALDI-TOF MS.
    Materials and methods
    γ-tubulin is an evolutionary conserved cytoskeletal protein, which plays essential roles in microtubule organization and nucleation . The subcellular distribution of this protein has been previously analyzed in a large variety of model organisms ranging from through and mammals. In some of these studies, γ-tubulin was detected only in the centrosomes-related organelles during both the interphase and mitosis . Other reports Protease Inhibitor Library revealed the expansion of γ-tubulin during the mitosis within the nearly entire mitotic spindle . To our knowledge, the distribution of γ-tubulin has not yet been analyzed in the zebrafish adult somatic cells. Previous studies revealed that the zebrafish heart regeneration depends on the proliferation of adult cardiomyocytes . Here, to understand the mitotic mechanisms associated with zebrafish heart regeneration, we analyzed the expression of both γ-tubulin and phosphohistone H3 (PH3), which demarcates the condensed chromosomes during the nuclear division. Our data provide evidence for the dynamic γ-tubulin expression during the cell cycle. To distinguish between the interphase/G0 and mitosis, we used phospho-(Ser10)-histone H3 (PH3) immunolabeling that demarcates the condensed chromosomes. To identify cardiomyocytes among other cell types in the heart, we used a transgenic fish line expressing EGFP under a cardiac specific promoter (::), and we performed anti-GFP immunostaining (A-C; ). Analysis of multiple heart sections revealed that in the non-mitotic zebrafish cardiomyocytes, γ-tubulin expression in restricted to a single spot in the vicinity of each nucleus, which corresponds to the centrosome . By contrast, all of the PH3-positive cardiac Protease Inhibitor Library were characterized by an expanded and stronger presence of γ-tubulin that was associated with the condensed chromosomes (=17 cells, 5 hearts) (C and E; C and D). Analysis of the red fluorescence with the same image adjustments revealed that this centrosomal pattern of γ-tubulin expression does not derive from background enhancement (). Thus, γ-tubulin is not restricted only to the duplicated centrosomes of the dividing cells, but it covers other domains of the mitotic spindle. Due to the high difference in the intensity of γ-tubulin signals between mitotic and non-mitotic cells, it was not possible to simultaneously display both types of expression patterns on the same image. The original confocal data had to be adjusted using Adobe Photoshop to visualize both aspects of γ-tubulin expression in the separate images of the same original specimen (D and F; C′and D′). Our analyses of mitotic cytoskeletal proteins will be helpful to understand the cellular mechanisms underlying the proliferative capacity of adult zebrafish cardiomyocytes. Experimental design, materials and methods
    Acknowledgments We thank V. Zimmermann for excellent technical assistance and for fish care and F. Chablais for specimens. This work was supported by the Swiss National Science Foundation, Grant numbers: 310030_138062 and CRSII3_147675 and Schweizerische Stiftung für die Erforschung der Muskelkrankheiten.
    Experimental design, materials and methods Rat, mouse, and human liver microsomes or S9 fractions (0.6mg protein, n=2) were solubilized in 2% SDS solution (1:1v/v ratio) and then diluted with 0.1M ammonium bicarbonate (pH 8.5) prior to reductive alkylation with dithiothreitol (2.5mM) and iodoacetamide (5mM). Additional ammonium bicarbonate (for trypsin), or 0.2% trifluoroacetic acid in 20% methanol (for pepsin), was added for an overnight digestion at a 1:50 (w/w) enzyme/protein ratio. Digests were neutralized, diluted with water, and subjected to solid-phase extraction (SPE) on a 1cm3 (30mg) OASIS HLB cartridge (Waters, Milford, MA), eluting with 100% methanol (1ml). Eluates were evaporated to dryness under vacuum, reconstituted in SCX buffer A (see below), and injected (100μl, 0.5mg protein) onto a Zorbax 300-SCX 150×2.1mm column with 5μm (300Å) particles (Agilent Technologies, Palo Alto, CA) using an Agilent 1200 series HPLC equipped with a binary pump, degasser, diode array detector and fraction collector. SCX fractionation was performed (250μl/min) with a gradient of 0–50% B in 15min, up to 100% B at 25min, then held for an additional 5min at 100% B, where buffers A and B were 10mM potassium dihydrogen phosphate in 25% acetonitrile (pH 2.75), and 1M potassium chloride in buffer A (pH 2.75), respectively. UV absorbance was monitored at 220 and 280nm. For trypsin samples, 3min (0.75ml) fractions were aliquoted into 1.5ml tubes between 1.5 and 19.5min, while for pepsin, 4min (1.0ml) fractions were collected between 1.5 and 25.5min. Fractions were evaporated to dryness under vacuum and kept at −30°C.