Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: Phosphomimetic but not SPM defective N-Spc110 proteins induce oligomerization of γ-TuSC. ( A ) Overlapped gel filtration chromatograms with molecular weight markers. The peak of the void-volume (V 0 , boxed area) corresponds to molecular weight fractions higher than 5000 kDa. Note that the concentrations of γ-TuSC and Spc110 1–220 variants in ( A ) were double as high compared to ( B ) and ( C ). ( B ) Gel filtration chromatograms of γ-TuSC incubated with phosphomimetic or non-phosphorylatable Spc110 1–220 mutant proteins. γ-TuSC was incubated in TB150 buffer with Spc110 1–220 proteins from Figure 2—figure supplement 1A . Complexes were analyzed by Superose 6 10/300 chromatography. Fractions corresponding to the peak were analyzed by SDS-PAGE and silver staining. ( C ) As ( B ) but with Spc110 1–220 variants that have defective SPM due to T18D or T18A. DOI: http://dx.doi.org/10.7554/eLife.02208.008

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: Filtration, Molecular Weight, Incubation, Mutagenesis, Chromatography, SDS Page, Silver Staining

Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: γ-TuSC does not oligomerize in TB150 buffer. ( A – C ) Purified γ-TuSC ( A ) spontaneously oligomerizes in BRB80 buffer ( B ) but not in TB150 buffer ( C ). Proteins were analyzed by Superose 6 10/300 and subsequently gel filtration fractions were subjected to SDS-PAGE and silver staining. Samples in the peak fraction were subjected to negative staining and protein complexes were analyzed by electron microscopy. Corresponding scale bars are shown on the images. DOI: http://dx.doi.org/10.7554/eLife.02208.005

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: Purification, Filtration, SDS Page, Silver Staining, Negative Staining, Electron Microscopy

Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: γ-TuSC Spc98Δ1–156 maintains self-oligomerization capability in BRB80 buffer. ( A ) γ-TuSC Spc98Δ1–177 was purified from insect cells, incubated in TB150 buffer, and then analyzed by Superose 6 10/300. The left panel presents the Coomassie Blue stained SDS-gel of purified γ -TuSC Spc98Δ1–177 . ( B ) γ-TuSC Spc98Δ1–177 in fraction 7 from ( A ) were subjected to negative staining and analyzed with electron microscopy. The particles showed irregular morphology, suggesting the formation of protein aggregates. Corresponding scale bars are shown in the images. ( C ) Similar experiment as performed in ( A ). Purified γ-TuSC Spc98Δ1–156 was incubated in TB150 or BRB80 buffer and then analyzed by Superose 6 10/300. γ-TuSC Spc98Δ1–156 oligomerized in BRB80 but not in TB150 buffer. ( D and E ) Similar experiment as performed in ( B ). After the gel filtration shown in ( C ), the peak fraction of γ-TuSC Spc98Δ1–156 (fraction 7 in BRB80 and fraction 13 in TB150) was subjected to negative staining and analyzed with electron microscopy. Corresponding scale bars are shown on the images. DOI: http://dx.doi.org/10.7554/eLife.02208.021

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: Purification, Incubation, Staining, SDS-Gel, Negative Staining, Electron Microscopy, Filtration

Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: Phosphorylation of T18 most likely affects γ -TuSC oligomerization promoting activity of Spc110 in a negative manner. ( A ) Phospho-specific antibody against phospho-T18 (pT18) was generated from guinea pigs. In vitro kinase assays were performed in the presence of Cdk1 as1 and Spc110 1–220 , either wild type (WT) or non-phosphorylatable Spc110 1–220-3A (Cdk1) (T18A-S36A-S91A). In vitro phosphorylated Spc110 1–220 was subjected to SDS-PAGE and immunoblot with the phospho-specific antibody. As negative control of kinase activity, Cdk1 as1 -Clb2 and Cdk1 as1 -Clb5 overexpressed and purified from budding yeast were inactivated with 1NM-PP1. Note that the same Spc110 1–220 samples were analyzed in Figure 5A with the anti-pS36-pS91 antibody. The Spc110 1–220 blot on the bottom is the same as in Figure 5A . ( B ) Quantification of tryptic phospho-T18 peptide (NMEFpTPVGFIK). Data were acquired in a data independent fashion in a pseudo MRM experiment in the Orbitrap mass spectrometer fragmenting peptides with m/z = 689.81. Extracted ion chromatogram of phosphorylated NMEFTPVGFIK was quantified. Expected retention time of the tryptic phospho-T18 peptide is 24.7 min. ( C ) Mass spectrum of fragmented phospho-T18 peptide. The graph summarizes the identified fragmented ions. ( D ) Gel filtration chromatograms of γ-TuSC. Spc110 1–220-5D was incubated with Cdk1 as1 -Clb2 in the presence (+1NM-PP1) and absence of the Cdk1 as1 inhibitor 1NM-PP1 and ATP. This allowed Spc110 T18 phosphorylation in the absence but not in the presence of 1NM-PP1. Adding 1NM-PP1 stopped all Cdk1 as1 kinase reactions. The so modified Spc110 1–220-5D-pT18 was then incubated with γ-TuSC. γ-TuSC was analysed by gel filtration using a Superose 6 column for oligomerization. The gel filtration fractions were analyzed by immunoblotting for Spc97/Spc98, Tub4, Spc110 1–220 , and Spc110 1–220−pT18 distribution. The anti-pT18 blot on the bottom is a 25-times of contrast enhancement of the blot on top. Fractions covering the void-volume peak were marked with a red rectangle outline. ( E ) Quantification of void-volume fractions shown in ( D ). Spc110 1–220-5D-T18D was included as control. Indicated protein band intensities of fractions covering the void-volume peak (red rectangle outline in ( D )) were summed and divided by the total intensity of fractions. * marks statistical significance at p

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: Activity Assay, Generated, In Vitro, SDS Page, Negative Control, Purification, Mass Spectrometry, Filtration, Incubation, Modification

Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: Phenotype of SPC110 mutants. ( A ) There was no significant difference in the protein levels of Spc110 variants. Asynchronous cells grown to OD 600 0.5 were harvested and subjected to TCA extraction for whole cell protein lysates. SDS-PAGE and immunoblotting with anti-Spc110 were performed. Tub2 was loading control. ( B ) The representative images of wild type SPC110 (WT) and spc110 cells in G1 (no bud) and S phase (small bud). Microtubules were marked with GFP-Tub1 and SPBs were marked with Spc42-mCherry. See Figure 6B for experimental set-up. BF: bright field. Scale bar: 4.5 µm. ( C ) The indicated wild type SPC110 (WT) and spc110 cells were grown at 37°C. The cell cycle stages were determined according to cell morphology and spindle length. Unfixed cells were analyzed for Spc97-GFP signal at SPBs. N = 30 for the number of cells analyzed for each category. * marks statistical significance at p

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: SDS Page

Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: Phosphorylation of N-Spc110 regulates the affinity to γ-TuSC. ( A ) GST pull-down assays were performed between γ-TuSC (containing His-tagged Spc97-6His and Spc98-6His) and the GST-tagged Spc110 1–220 proteins. The bound proteins were eluted with sample buffer and separated on SDS-PAGE and analyzed by immunoblotting with anti-His, anti-GST, and anti-Tub4 antibodies. Infrared-dye-labelled secondary antibodies were applied and detected with Li-cor imaging system. ( B ) Quantification for bound Spc97, Spc98, and Tub4 from ( A ) at 300 nM Spc110 1–220 . ** marks statistical significance at p

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: SDS Page, Imaging

Journal: eLife

Article Title: Cell-cycle dependent phosphorylation of yeast pericentrin regulates ?-TuSC-mediated microtubule nucleation

doi: 10.7554/eLife.02208

Figure Lengend Snippet: Purification of Spc110 1–200 variants. ( A ) Purified GST-Spc110 1–220 variants. ∼1 μg of protein was loaded to each lane. The SDS-PAGE was stained with Coomassie Blue. ( B ) Calibration of Superose 6 10/300 gel filtration column. Following protein markers were used: thyroglobulin (667 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa). Partition coefficient (K av ) was calculated using the equation: (V e –V 0 )/(V c –V 0 ), where V 0 = column void volume, V e = elution volume, and V c = geometric column volume. The calibration curve (bottom) of K av vs log molecular weight was plotted in semi-logarithmic scale. ( C ) Gel filtration chromatograms of purified GST-Spc110 1–220 variants. Proteins were analyzed in TB150 buffer by Superose 6 10/300 chromatography. DOI: http://dx.doi.org/10.7554/eLife.02208.007

Article Snippet: The reaction was stopped by the addition of SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen, Carlsbad, CA), and autoradiography.

Techniques: Purification, SDS Page, Staining, Filtration, Molecular Weight, Chromatography

Journal: Scientific Reports

Article Title: Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

doi: 10.1038/srep36714

Figure Lengend Snippet: Kap60 and Yar1 compete for Rps3 binding. ( a ) Kap60 co-elutes after Rps3-TAP purification. An RPS3 -TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A KAP60 -3xHA strain without TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. Note that there is some unspecific recovery of Kap60-3xHA in the eluate of the control strain; however, the protein is clearly enriched in the Rps3-TAP eluate. Kap123: Kap123 was detected in this band by mass spectrometry (Mascot score 211, 11 matched peptides, 12% sequence coverage). Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 85, 4 matched peptides, 10% sequence coverage). ( b ) GST-tagged importins (Kap60ΔIBB or Kap123) were immobilized on glutathione-agarose beads. Truncated Kap60 lacking the N-terminal IBB domain (80 amino acids) was used in order to prevent inhibition of cargo binding by this domain in the absence of Kap95. Beads were incubated with purified, recombinant His6-Rps3/Flag-Yar1 complex or purified Flag-Yar1. Importins and bound material were eluted by boiling. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting with indicated antibodies. Asterisks in the Coomassie stained gel mark the respective importins, circles indicate bound Rps3. ( c ) Disruption of the Rps3-NLS impairs Kap60 binding. GST-tagged Kap60ΔIBB was incubated with His6-Rps3/Flag-Yar1 complex containing wild-type Rps3 or Rps3(K7/K10 > A) mutant protein. Kap60ΔIBB was eluted by TEV-cleavage. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting.

Article Snippet: Eluates were analyzed on 12% or 4–12% SDS-polyacrylamide gels (Invitrogen) and Coomassie staining or Western blotting.

Techniques: Binding Assay, Purification, Negative Control, SDS Page, Staining, Western Blot, Mass Spectrometry, Sequencing, Inhibition, Incubation, Recombinant, Mutagenesis

Journal: Scientific Reports

Article Title: Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

doi: 10.1038/srep36714

Figure Lengend Snippet: Yar1 and Kap60 are genetically and physically linked. (a) Growth defect of a yar1 deletion strain is enhanced in combination with kap60- ts and kap95- ts mutant alleles. kap60- ts/ yar1 Δ or kap95- ts/ yar1 Δ strains were transformed with plasmids harboring the indicated wild-type alleles or empty plasmids (−). Cells were spotted in 10-fold serial dilutions on SD-Leu-Trp plates and incubated at the indicated temperatures. ( b ) Kap60 is co-purified with Yar1-TAP. A YAR1- TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A strain containing KAP60 -3xHA but no TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 255, 5 matched peptides, 15% sequence coverage).

Article Snippet: Eluates were analyzed on 12% or 4–12% SDS-polyacrylamide gels (Invitrogen) and Coomassie staining or Western blotting.

Techniques: Mutagenesis, Transformation Assay, Incubation, Purification, Negative Control, SDS Page, Staining, Western Blot, Mass Spectrometry, Sequencing

Journal: Scientific Reports

Article Title: Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

doi: 10.1038/srep36714

Figure Lengend Snippet: Kap60/Kap95 orchestrate nuclear import of the Rps3/Rps3/Yar1 complex. ( a ) Kap60/Kap95 are bound to the Rps3/Yar1 complex in vivo . The cell lysate of a YAR1 -TAP, RPS3 -Flag strain was subjected to affinity purification via the protein A tag of Yar1. The obtained TEV (tobacco etch virus) eluate was further purified in a second affinity purification step via Rps3-Flag. As negative control the lysate of an untagged wild-type strain was subjected to the same purification procedure. Lysates and final eluates (E-Flag) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. ( b ) Kap60 binds the Rps3/Yar1 complex in vitro . His6-Rps3/Flag-Yar1 complex was immobilized via His6-Rps3 on Ni-NTA-agarose beads and incubated with increasing amounts of purified Kap60ΔIBB. After elution of His6-Rps3 (Ni-NTA-eluate), bound material was further subjected to Flag-purification via Flag-Yar1 (Flag-eluate). As negative control, empty Ni-NTA agarose or Flag-agarose beads were incubated with the highest Kap60ΔIBB concentration. The eluates were analyzed by SDS-PAGE and Coomassie staining. ( c ) Model describing the formation of the Yar1/Rps3/Rps3/Kap60/Kap95 import-complex that allows simultaneous targeting of an Rps3 dimer bound to one Yar1 chaperone into the nucleus.

Article Snippet: Eluates were analyzed on 12% or 4–12% SDS-polyacrylamide gels (Invitrogen) and Coomassie staining or Western blotting.

Techniques: In Vivo, Affinity Purification, Purification, Negative Control, SDS Page, Staining, Western Blot, In Vitro, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: Sequence Analysis of the IL28A/IL28B Inverted Gene Duplication That Contains Polymorphisms Associated with Treatment Response in Hepatitis C Patients

doi: 10.1371/journal.pone.0029983

Figure Lengend Snippet: Allelic discrimination plot for the rs12979860 genotyping assay. Allelic discrimination plot generated by the SDS 2.3 software (Applied Biosystems Inc.) for the rs12979860 genotyping assay using the 48 sample DNA panel. Red and blue circles represent the two homozygous genotypes (T/T and C/C), the green circles represent heterozygous genotype (C/T) and the black squares represent no template control.

Article Snippet: Plates were read on an ABI 7900HT (Applied Biosystems Inc.) and the data was analyzed using SDS 2.3 software (Applied Biosystems Inc.).

Techniques: Genotyping Assay, Generated, Software