Enrichment of salt-soluble PPP complexes from the mitotic spindle proteome via MC-Sepharose affinity chromatography.

<p>The mitotic spindle and chromatin associated proteome was enriched and separated into soluble proteins and MTs according to (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone-0039510-g001" target="_blank">Fig. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s002" target="_blank">2</a>). Soluble proteins were brought to 420 mM NaCl and PPP complexes isolated via affinity chromatography with MC-Sepharose and Tris-Sepharose as a control matrix. Matrices were washed with buffer A at low (<b>A</b>, 300 mM NaCl) or medium (<b>B</b>, 500 mM NaCl) ionic strength. Proteins were eluted with 1% SDS, concentrated, separated via SDS-PAGE and visualized by colloidal silver stain (<b>A</b>, <b>B</b>). Specific interactors (<b>B</b>, bands A–D) were isolated and identified via LC-MS/MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s003" target="_blank">Fig. S3</a>). <b>C</b>. Western blot analyses with the respective antibodies to verify the specific presence of the interactors identified in <b>B</b>.</p

Proteomic screen in the simple metazoan identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Casignalling-3

<p><b>Copyright information:</b></p><p>Taken from "Proteomic screen in the simple metazoan identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Casignalling"</p><p>http://www.biomedcentral.com/1471-2121/8/31</p><p>BMC Cell Biology 2007;8():31-31.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC1964759.</p><p></p>nexin1 (lanes 1-6). Inputs of 14-3-3 HyA (lane 1) and 14-3-3 HyB (lane 4) represent 12.5% of the total 14-3-3 proteins used in each assay. Eluted 14-3-3 HyA (lanes 2 and 3) and 14-3-3 HyB (lanes 5 and 6) are shown after pulldown with GST-fusion-proteins. Western blots were probed with anti-Xpress antibody, which recognises the His-tag of the bacterially expressed 14-3-3 proteins. In (B) Western blots with samples from (A) were probed with anti-GST antibody to show that equal amounts of GST-fusion-protein were eluted in all assays. Asterisks indicate full-length GST-fusion proteins. Smaller fragments represent degradation products

Toposome members as part of the mitotic phosphatome.

<p>Mitotic spindles were isolated (fraction 1-3) and MAPs and MTs separated as before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s001" target="_blank">Fig. S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s002" target="_blank">2</a>). Diluted MAPs (IN) were subjected to MC- or Tris-Sepharose affinity chromatography and proteins eluted with 1% SDS (MC, Tris respectively). Each eluate had a small precipitate after concentration which was loaded separately (*). Western blot analyses were performed with antibodies against indicated proteins.</p

Pull down of purified, bacterially expressed PP1 isoforms via His-tagged RNA Helicase Ddx21.

<p>Bacterially expressed and purified PP1 (α, β, γ) and His6-Ddx21 (wt, motif1, motif2, double) were incubated together and then mixed with precleared Ni-NTA beads, all at 4°C. Each PP1 isoform was also incubated without Ddx21 (-) to verify unspecific binding of PP1 to the Ni-NTA beads. Enriched His6-Ddx21 and co-precipitated PP1 isoforms were washed and boiled in 2 X SDS-PAGE sample buffers. PP1 inputs, Ddx21 eluates and negative control (-) eluates were separated and analysed by western blot for the presence of Ddx21 and PP1.</p

Proteomic screen in the simple metazoan identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Casignalling-4

<p><b>Copyright information:</b></p><p>Taken from "Proteomic screen in the simple metazoan identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Casignalling"</p><p>http://www.biomedcentral.com/1471-2121/8/31</p><p>BMC Cell Biology 2007;8():31-31.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC1964759.</p><p></p>tags from at NCBI. Helices 1-9 are indicated. Conserved amino acids that have been shown in the crystal structure to constitute the binding groove for target proteins are indicated with asterisks (basic face of the groove) and circles (hydrophobic surface) above the sequence (information from [24]). Alignments were made with the program GeneJockeyII (under MacOS9

Ddx21 interacts with PP1 directly and influences PP1α activity.

<p><b>A.</b> Bacterial expression & purification of Ddx21 and PP1. HIS6-Ddx21 was purified from DH5α or BL21-DE3* expressing cells with SP-Sepharose and Ni²⁺-NTA. PP1 isoforms were purified according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510-Moorhead3" target="_blank">[66]</a>. Purified Ddx21 (*) and PP1 (*) isoforms were separated via SDS-PAGE and stained with colloidal. <b>B.</b> PP1 isoform activity assay in the presence of Ddx21. Proteins were premixed according to increasing molar ratios of Ddx21:PP1 and then incubated with PP1 substrate (pNPP). Data are shown as mean ± S.D. (n = 3). PP1 activity is normalized against activity without Ddx21 (point 0 on X-axis, normalized to 1). <b>C.</b> Direct interaction between PP1 and Ddx21 occurs partially through the canonical PP1 binding motif. His6-Ddx21 (wt); His6-Ddx21_RARA (aa202–208: motif 1); (aa440–444: motif2) and (double) were expressed in and purified from DH5α cells. Proteins were separated via SDS-PAGE and loaded amounts verified by colloidal stain (loading control). Purified PP1α, β and γ isoforms were labelled with DIG and overlay assays were executed and visualised with α-DIG antibodies.</p

PP1 is a toposome interaction partner within the mitotic spindle proteome. A.

<p>Immunoprecipitation of SRPK1 identifies toposome components and PP1<b>.</b> Mitotic spindles were isolated and MAPs separated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s001" target="_blank">Fig. S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone.0039510.s002" target="_blank">2</a>. Diluted MAPs (IN) were subjected to immunoprecipitations with SRPK1 or control (mouse IgG) antibodies. Bound proteins were eluted by boiling in 1X sample buffer. Western blot analyses were performed with antibodies against indicated proteins. <b>B.</b> PP1α, γ are the preferred interaction partners for SRPK1. Experiments were carried out as in <b>A</b>, except for the use of antibodies against human PP1 isoforms or control antibodies (PIS rabbit IgGs).</p

Proteomic screen in the simple metazoan identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Casignalling-1

<p><b>Copyright information:</b></p><p>Taken from "Proteomic screen in the simple metazoan identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Casignalling"</p><p>http://www.biomedcentral.com/1471-2121/8/31</p><p>BMC Cell Biology 2007;8():31-31.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC1964759.</p><p></p

Insulin induces binding of 14-3-3 to endogenous IRS-2 and phosphorylates Ser-573 on IRS-2.

<p><i>A.</i> Fao cells were starved for serum overnight and incubated for 30 min with either 10 nM insulin or 50 ng/ml IGF-1. 250 µg protein was immunoprecipitated with IRS-2 antibody and separated on a 5–15% gradient gel. Overlay assay followed stripping and reprobing with IRS-2 antibody as loading control. Successful stimulation is shown as phosphorylation of p-Thr-308 and corresponding Akt/PKB reblot. <i>B.</i> Fao cells were starved for serum overnight and stimulated with 10 nM insulin for the indicated time points. 100 µg of protein was separated on a 7.5% gel and membranes were probed with specific antibodies against p-Ser-573 of IRS-2 and p-Thr-308 of Akt/PKB. For loading control membranes were stripped and reprobed with protein antibody.</p

Peptide displacement partially releases mitotic Ddx21 from the MC-Sepharose matrix. A.

<p>Prp8 and Ddx21 enrich in the mitotic spindle proteome. Mitotic spindle and associated fractions were obtained and proteins loaded and separated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039510#pone-0039510-g001" target="_blank">Fig 1</a>. Western blot analyses were with antibodies against Ddx21 and Prp8. <b>B.</b> A fraction of Ddx21 is displaced by an excess of a PP1-binding peptide. MAPs were incubated with MC- or Tris-Sepharose matrices and PP1-interaction partners eluted sequentially with an excess of a scrambled PP1-binding peptide (not shown) followed by a PP1-binding peptide (-RVRW-). Any remaining PPPs, including PP1, and their interaction partners were finally eluted with 1% SDS. Proteins were separated via SDS-PAGE and presence of Ddx21, Prp8 and PP1 verified by western blot analyses with the respective antibodies.</p