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  • br Acknowledgments This work was supported

    2018-11-07


    Acknowledgments This work was supported in part by grants from the Mary Kirkland Center for Lupus Research; Colleck Research Fund, Weill-Cornell Medical College; and the Colgate-Palmolive, Co. to AAS.
    Specifications table Value of the data Data, experimental design, materials and methods Applying a quantitative shotgun proteomics approach (One-dimensional nLC-ESI-MS/MS) we have obtained insight on the proteome composition of the haloarchaeon H. volcanii H26 wt vs a conditional mutant that synthesizes suboptimal amounts of the membrane protease LonB (HVLON3). Supplementary Table S1 shows a list of all the proteins that were identified combining these strains, organized according to their functional category. A total of 1778 proteins were detected (including membrane and cytoplasm fractions) representing 44% of the predicted H. volcanii theoretical proteome. Additionally, this data allowed the identification of the unique proteins detected in each growth phase and/or strain ( Supplementary Table S2). Supplementary Table S3 (1–4) shows the Proteome Discoverer database search results for all the replicates (4) of the wt, HVLON3 and HVABI strains.
    Specifications table Value of the data Data, materials and methods AMPK serves as a master metabolic regulator in eukaryotic cells [2]. Moreover, AMPK is also critical to other cellular functions, such as the stress response [3]. The formation of cytoplasmic SGs is one of the hallmark responses to many types of stress [4]. Here, we used different neurokinin receptor antagonist (Fig. 1) and a distinct cell line from another organism (Fig. 2) to verify the isoform-specific recruitment of the AMPK-α subunit to SGs. In addition to the catalytic α subunits, we assessed the interaction of regulatory β and γ AMPK subunits with SGs upon treatment with arsenite or diethyl maleate (DEM), components that induce oxidative stress (Figs. 3–5). AMPK-α knockdown changed SG parameters [1], and we tested whether this can be linked to the abundance of SG marker proteins TIA-1/TIAR, G3BP1 and HuR. To this end, we quantified the levels of core SG proteins under control and AMPK-α knockdown conditions. The knockdown of AMPK-α1 or α2 did not cause significant changes in the concentration of these three SG marker proteins (Fig. 6).
    Acknowledgments This work was supported by grants from NSERC (155509-2009), FQRNT, Quebec and HSFC. HM is a recipient of a doctoral fellowship from NSERC. We are grateful to Drs. J. Orlowski and R. Jones (McGill University) for their generous gift of reagents.
    Specifications Table Value of the data Experimental design, materials and methods Fig. 1 shows the general workflow of the Phospho-iTRAQ approach. (A) Experimental workflow: in the “in-solution” approach (left panel), the soluble protein extracts (R1) of control and EGF-stimulated HeLa cells were spiked with an internal peptide standard (IS2) of heavy phosphopeptides and compared by Phospho-iTRAQ. When only one sample is mined (EGF), the flexibility of the protocol allows for gel purification and fractionation and thus for complementing the soluble protein extract (R1) with the hydrophobic fraction diluted in strongly denaturing buffer (R3) to increase the number of annotated phosphopeptides (“in-gel” approach, right panel). The EGF-stimulated cells were spiked with a phosphoprotein internal standard (IS1) before fractionation on a 1D PAGE into four molecular weight fractions followed by Phospho-iTRAQ. (B) PhosphoiTRAQ protocol: a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Afterward, samples are immediately recombined and split into three parts for the LC–MS/MS analysis on three different instruments. (C) Data Analysis: raw data was processed by the respective vendor׳s software, and MGF-files were searched against the SwissProt Human database using Mascot. Exported DAT-files were imported into Rover for ranking of the iTRAQ ratios and were further analyzed in Excel. (D) Data: initially phosphorylated peptides have skewed iTRAQ ratios and arise out of the center of the log-normal distribution of the whole precursor population. The mean of the log-normal ratio distribution is located around zero since the vast majority of the peptides in the data have equal 114/115 or 116/117 reporters.