Cohesion policy funding for innovation and the knowledge economy

This paper examines roles of cohesion policy in financing support for research and development and technological innovation

Impaired BAT3 recruitment to Derlin2 slows dislocation.

<p>A. 293T cells were transiently transfected with TCRα and either pcDNA, Derlin2, or Derlin2-GFP (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028542#pone-0028542-g001" target="_blank">figure 1D</a>). TCR degradation was assessed by pulse-chase analysis. B. Densitometric quantitation of the relative amount of protein is shown (n = 4).</p

BAT3 associates with Derlin2.

<p>A. HA-Ri332 was synthesized in a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomal membranes (MM). Following NP40-mediated lysis, Ri332 was retrieved by immunoprecipitation. The immunoprecipitate and input samples were blotted for either BAT3 or HA as indicated. B. 293T cells were subjected to NP40 lysis, followed by retrieval of the indicated proteins. Pre-immune serum served as a control. The eluates were blotted for BAT3, as were the input control samples. C. Immunofluorescence of Hela cells using antibodies against endogenous BAT3 (green) and Derlin2 (red). Scale bar = 5 µm. D. 293T cells were transiently transfected with the indicated constructs, subjected to NP40 lysis followed by an immunoprecipitation for Derlin2. Immunoprecipitates were blotted for BAT3. Input samples were blotted for BAT3 and Derlin2. The <i>asterisk</i> indicates non-specific polypeptides.</p

BAT3 is required for dislocation of TCRα.

<p>A. 293T were stably transduced with short RNA hairpins against either GFP or BAT3. SDS lysates were immunoblotted for either BAT3 or p97. B. BAT3 knock-down cells (a) were transiently transfected with HA-Ri332 and subjected to pulse-chase analysis. Densitometric quantitation of the relative amount of protein is shown (n = 9). C. As in (b), except that cells were transfected with TCRα (n = 3).</p

TCRα is engaged by BAT3.

<p>293T cells were transiently transfected with TCRα and either empty vector, UBX-EBV, or p97 QQ, and labeled overnight with [³⁵S] methionine/cysteine to achieve steady state labeling. Cells were harvested and subjected to NP40 lysis. The lysates were precleared using pre-immune serum and immobilized protein A. Lysates were adjusted for total levels of incorporated isotope and subjected to immunoprecipitation for TCRα. The captured protein was eluted in 1% SDS at 37°C followed by a second immunoprecipitation for the indicated proteins.</p

BAT3 localizes to a complex with Derlin2 and Ri332.

<p>Hela cells were plated on coverslips and transiently transfected with HA-Ri332 and empty vector (A), YOD1 C160S (B) or UBX-EBV (C). After paraformaldehyde fixation, cells were labeled for HA (green), BAT3 (red), and Derlin2 (blue). Scale bar = 5 µm.</p

Hepta-Mutant Staphylococcus aureus Sortase A (SrtA_7m) as a Tool for <i>in Vivo</i> Protein Labeling in Caenorhabditis elegans

<i>In vivo</i> protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA_7m) expressed in C. elegans is functional and supports <i>in vitro</i> sortase reactions in a low-Ca²⁺ environment. Feeding SrtA_7m-expressing C. elegans with small peptide-based probes such as (Gly)₃- biotin or (Gly)₃-fluorophores enables <i>in vivo</i> target protein modification. SrtA_7m also catalyzes the circularization of suitably modified linear target proteins <i>in vivo</i> and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve <i>in vivo</i> protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins <i>in vivo</i>

Hepta-Mutant Staphylococcus aureus Sortase A (SrtA_7m) as a Tool for <i>in Vivo</i> Protein Labeling in Caenorhabditis elegans

<i>In vivo</i> protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA_7m) expressed in C. elegans is functional and supports <i>in vitro</i> sortase reactions in a low-Ca²⁺ environment. Feeding SrtA_7m-expressing C. elegans with small peptide-based probes such as (Gly)₃- biotin or (Gly)₃-fluorophores enables <i>in vivo</i> target protein modification. SrtA_7m also catalyzes the circularization of suitably modified linear target proteins <i>in vivo</i> and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve <i>in vivo</i> protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins <i>in vivo</i>

Hepta-Mutant Staphylococcus aureus Sortase A (SrtA_7m) as a Tool for <i>in Vivo</i> Protein Labeling in Caenorhabditis elegans

<i>In vivo</i> protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA_7m) expressed in C. elegans is functional and supports <i>in vitro</i> sortase reactions in a low-Ca²⁺ environment. Feeding SrtA_7m-expressing C. elegans with small peptide-based probes such as (Gly)₃- biotin or (Gly)₃-fluorophores enables <i>in vivo</i> target protein modification. SrtA_7m also catalyzes the circularization of suitably modified linear target proteins <i>in vivo</i> and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve <i>in vivo</i> protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins <i>in vivo</i>

Site-specifically labeled HA-Srt protein is incorporated into virions.

<p>(<b>A</b>) <b>Experimental setup.</b> Confluent monolayers of MDCK cells were infected with an MOI = 0.5 during 4.5 hours after which cells were starved and pulse-labeled with [^S35]Cysteine/Methionine for 20 minutes. After a 2 hour chase, a second pulse-labeling was performed using 100 µM sortase and 250 µM biotin probe to label surface accessible HA-Srt. At indicated timepoints, both cell supernatant as well as cell lysate was analyzed for presence of viral proteins. (<b>B</b>) <b>Surface behavior HA on infected MDCK cells analyzed via affinity adsorption to neutravidin-agarose.</b> At indicated timepoint, cells were lysed in 0.5% NP40 buffer and biotin labeled HA-Srt remaining at the cell surface recovered via affinity adsorption on neutravidin-agarose. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. (<b>C</b>) <b>Accumulation of HA-biotin in supernatant analyzed by affinity adsorption to neutravidin-agarose.</b> Accumulation of biotin-HA-Srt in the supernatant of the cells analyzed in 4B was measured via immunoprecipitation on neutravidin-agarose. Supernatant was lysed via addition of NP40 buffer prior to biding to beads. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. (<b>D</b>) <b>Quantification of HA loss from the cell surface.</b> Densitometric quantification of radioactivity was performed on autoradiographs from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002604#ppat-1002604-g004" target="_blank">figure 4B and 4C</a>. Total levels of HA-Srt were quantified relative to the levels at the cell surface at t = 0 hrs (top graph). To quantify the kinetics of budding, loss of HA-Srt from the cell surface was quantified as percent reduction relative to the t = 0 timepoint at the cell surface. The rate of accumulation in the cell supernatant was quantified relative to the maximal amount recovered at the t = 10 hrs timepoint. (<b>E</b>) <b>Accumulation of whole virus particles analyzed by affinity adsorption to chicken erythrocytes.</b> Accumulation of complete virus particles in the cell supernatant was measured via affinity adsorption on chicken erythrocytes. Supernatant from cells analyzed in 4B was removed at indicated timepoints and mixed with chicken erythrocytes for 30 minutes at 4°C. Cells and bound viral particles were lysed in 2× SDS sample buffer, proteins resolved on 12.5% SDS-PAGE and visualized via autoradiography. (<b>F</b>) <b>Kinetcs of virus accumulation as analyzed by adsorption to neutravidin-agarose versus erythrocytes.</b> Densitometric quantification of radioactivity was performed to compare the rate of HA-Srt accumulation in the supernatant compared to whole viral particles (4C versus 4E). Numbers were normalized at t = 1 hrs at which both methods were used.</p