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  • procyanidin b2 br Acknowledgments The CSGID project has

    2018-11-07


    Acknowledgments The CSGID project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contracts nos. HHSN272200700058C and HHSN272201200026C. We thank Sankar Krishnna for the human enolase 1 sample. The authors wish to thank members of the Structural Biology Center (SBC) at Argonne National Laboratory for their help with X-ray diffraction data collection. The operation of SBC beamlines is supported by the U.S. Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357.
    Specifications table
    Value of data
    Data The Affymetrix mRNA profile data are provided as CEL files deposited on GEO (GSE37212)(doi:10.1016/j.biochi.2014.09.021 [1]). RNA was extracted from miR-21 over-expressing Jurkat cells (pRRL-21) and matched control cell line (pRRL-Ctrl). For each cell line Input, AGO2 IP and isotype matched IgG IP samples were analysed.
    Experimental design, materials and methods
    Acknowledgements
    Here we report the in silico predictions data of the non-synonymous changes found in 41 HCM patients and in 3 HCM-related cases (). Experimental design, materials and methods
    Acknowledgments This work funded by the Department of Molecular Medicine, Sapienza University of Rome. This work was also partially funded by the Department of Biochemical Sciences Sapienza University of Rome (prot. C26A149EC4).
    Specifications Table
    Value of the data
    Data
    Experimental design, materials and methods NCALD was expressed in E. coli ER2566 as described in [1]. Briefly, cells grown overnight were inoculated (1% inoculum) into fresh LB medium and grown to an optical density of 0.6 at 600nm. IPTG (1mM final concentration) was then added for induction. For myristoylation, cells with yeast N-Myristoyl Transferase were used and myristic procyanidin b2 was supplemented. Cells without the transferase were used to generate the non-myristoylated form; the supplementation was also skipped. Cells were collected 2.5h after induction, sonicated and the protein was purified on phenyl sepharose columns as described previously [2,3]. The purified protein was then washed with calcium-depleted Tris–Cl (20mM; pH 7.5) to remove any residual calcium. Calcium removal was through the use of Chelex-100 resin (BioRad Laboratories, CA, USA) using standard procedures. For mass spectrometry, protein samples were co-crystallized with a 1:1 mixture of sinapinic acid and matrix solution (50% acetonitrile/0.05% trifluoroacetic acid in water). Mass spectrometric analyses were carried out in linear, negative modes on a Bruker LRF MALDI-TOF instrument. The mass values were in good agreement with those reported earlier for the respective forms [4] (see Table 1). There was no peak corresponding to the non-myristoylated form in the myristoylated preparation. Mobility Retardation of NCALD (and other NCS proteins) in native gels has been documented. The retardation was directly dependent on the concentration of calcium. In order to determine if myristoylation of NCALD was essential for the calcium-dependent mobility shift, analyses were carried out with the non-myristoylated (Myr−) or myristoylated (Myr+) NCALD. Protein was incubated in the presence of indicated concentration of calcium using calibration buffers and electrophoresed in native gels as described [1]. A representative image of the gel is presented in Panel A (Fig. 1). Relative mobility values were determined from at least three experiments and plotted as a function of calcium concentration (Fig. 1; Panel B). To facilitate direct comparison, the data for the myristoylated NCALD has been reproduced from [1].
    Acknowledgements The work was supported by grants from the New Jersey Health Foundation (previously UMDNJ) and the Osteopathic Heritage Foundation. The support by the RowanSOM Graduate School of Biomedical Sciences is also acknowledged. Dr. Michael Anikin is gratefully acknowledged for his help with mass spectrometry.