, MgCl2) supplemented with protease inhibitors (Aprotinin 2µg.mL-1 , Leupeptin 1µg.mL-1 , Pepstatin 1µg.mL-1 , PMSF 50µg.mL-1 ) then lysed using sonication for 4 minutes on ice, and centrifuged at 18000 rpm for 25 minutes at 4?C
, Clontech) preequilibrated in lysis buffer, and incubated for 2 hours in 50ml falcons on a rotator at 4 ?C. Beads were collected and washed with lysis buffer then transferred on Bio-Spin columns (Biorad) pre-washed in lysis buffer, Columns were further washed with lysis buffer followed by a wash buffer (1.5x PBS, 50mM Imidazole, 250mM NaCl, 0.1% Igepal, 10% Glycerol, 1mM MgCl2)
, Nmd4 was further dialyzed against calmodulin binding buffer (1x PBS, 100mM NaCl, 0.1% Igepal, 10% Glycerol, 1mM MgCl2, 4mM CaCl2, 1mM DTT) overnight at 4?C in Spectrapor-4, pp.12-14
, then mixed with 500?l Calmodulin Affinity Resin (= 1ml of 50% slurry, Agilent) preequilibrated in binding buffer, MWCO)
,
, Proteins were finally dialyzed against 1.5x PBS, 150mM NaCl, 10% (w/v) glycerol, 1mM DTT and 1mM MgCl2 in Spectrapor-4 (12-14 MWCO) then stored at-80?C
, In vitro pull-down assays
, Pull-down was performed using preblocked calmodulin affinity beads (Agilent)
, Preblocking beads. Briefly, in order to preblock beads, 1 ml calmodulin sepharose beads (50% Slurry) were spun, and resuspended in 20mM Hepes, 500mM NaCl, 0.1% Igepal, 0.08mg.ml-1 glycogen carrier, 0.08mg.ml-1 tRNA and 0.8mg.ml-1 BSA. After 2 hours at 4°C, beads were washed 3 times (20mM Hepes, 150mM NaCl, 0.1% Igepal) then resuspended in 500µl 1x binding buffer 250/10 (20mM Hepes
, Each mix contained 2µg of each protein in 30µl reaction mixes. NaCl and glycerol concentrations were adjusted to 150mM and 15% respectively. Five microliters (1/6) aliquots were REFERENCES Boratyn GM, vitro pull-down assay. Recombinant CBP-Nmd4, yeast Upf1 and human Upf1 proteins were thawed on ice, vol.7, p.12, 2012.
Saccharomyces Genome Database: the genomics resource of budding yeast, Nucleic Acids Res, vol.40, pp.700-705, 2012. ,
MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification, Nat Biotech, vol.26, pp.1367-1372, 2008. ,
Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment, J. Proteome Res, vol.10, pp.1794-1805, 2011. ,
STAR: ultrafast universal RNA-seq aligner, Bioinformatics, vol.29, pp.15-21, 2013. ,
Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase, PLoS One, vol.9, 2014. ,
A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae, Yeast, vol.13, pp.837-848, 1997. ,
Global analysis of protein expression in yeast, Nature, vol.425, pp.737-741, 2003. ,
Functional profiling of the Saccharomyces cerevisiae genome, Nature, vol.418, pp.387-391, 2002. ,
Rapid and reliable protein extraction from yeast, Yeast, vol.16, pp.857-860, 2000. ,
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biology, vol.15, p.550, 2014. ,
Comprehensive analysis of diverse ribonucleoprotein complexes, Aitchison JD & Rout MP, vol.4, pp.951-956, 2007. ,
MetaPhOrs: orthology and paralogy predictions from multiple phylogenetic evidence using a consistency-based confidence score, Nucleic Acids Res, vol.39, p.32, 2011. ,
The p21-activated protein kinase inhibitor Skb15 and its budding yeast homologue are 60S ribosome assembly factors, Mol. Cell. Biol, vol.27, pp.2897-2909, 2007. ,
URL : https://hal.archives-ouvertes.fr/pasteur-01404696
Absolute Quantification of Proteins by LCMSE A Virtue of Parallel ms Acquisition, Mol Cell Proteomics, vol.5, pp.144-156, 2006. ,
2016 update of the PRIDE database and its related tools, Nucleic Acids Res, vol.44, pp.447-456, 2016. ,
A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids, Anal. Biochem, vol.138, pp.141-143, 1984. ,