Size fractionation of complexes containing FLAG-CycC and myc-Cdk8.

<p><b>A</b>. 1×10⁹ vegetatively growing Ax2[<i>myc-cdk8</i>, FLAG-<i>cycC</i>] cells were harvested and the extracted nuclei lysed and subjected to gel filtration on a Superose 6 column. Fractions (7–18) were resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with α-FLAG and α-myc antibodies. Positions of molecular weight standards are specified above. <b>B</b>. 1×10⁹ Ax2[<i>myc-cdk8</i>] cells were harvested after either vegetative growth (V), development in shaking suspension for 8 hours with high (500 µM) cAMP added after 4 hours, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010543#pone-0010543-g002" target="_blank">Figure 2</a>, (S) or development on filters up to the first finger stage (F). A nuclear extract from each of these cells was fractionated by gel filtration on a Superose 6 column. Samples of fractions 7 to 19, as indicated, were resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an α-myc antibody. <b>C</b>. 2×10⁹ Ax2[<i>myc-cdk8</i>] cells were resuspended in KK₂ buffer and pulsed with 50 nM cAMP for 4 hrs. Cells were then split and either left untreated (−) or treated with 500 µM cAMP every hour (+) until the cells were harvested at 8 hrs. Nuclei were isolated, lysed and analysed by gel filtration chromatography and western blot as above.</p

Developmental expression of epitope-tagged cyclin C from its own promoter.

<p><b>A</b>. Exponentially growing cells overexpressing tagged cyclin C from its own promoter (Ax2[<i>cycC</i>::<i>cycC</i>-CTAP] cells) were harvested, washed and developed on filters at a density of 3×10⁶ cells/cm² and samples harvested for western and northern blots at the times indicated. Development was complete after 24 hours. Western samples were analysed on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an α-mouse IgG secondary antibody (as the TAP tag present in the protein contains an IgG binding domain) and an α-actin antibody to control for loading. RNA samples were resolved on a 1% formaldehyde gel, transferred to a nylon membrane and probed with a ³²P labelled fragment of the <i>cycC</i> gene and the <i>IG7</i> gene (to control for loading). The blot was exposed overnight to reveal cycC expression. B. Exponentially growing Ax2 cells were harvested and developed on filters as described above. RNA was isolated at the times shown and probed with a fragment of the cycC gene, The blot was exposed for 10 days. The levels of 17S and 26S rRNA are shown as a control for equal loading. <b>C</b>. Ax2[<i>cycC</i>::<i>cycC-</i>CTAP] cells were shaken in KK₂ buffer at 5×10⁶ cells/ml and pulsed with 50 nM cAMP every 5 mins. After 4 hrs cells were split and incubated in the presence (+) or absence (−) of 500 µM cAMP (added every hr for the duration of the experiment). Samples were taken at the time points indicated and analysed by western and northern blot as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010543#pone-0010543-g001" target="_blank">Figure 1</a>. <b>D</b>. Ax2[<i>cycC</i>::<i>cycC-</i>CTAP] cells were shaken in KK₂ buffer and pulsed with 50 nM cAMP as above. After 4 hrs the cells were split into three. One sample acted as an untreated control, one was pulsed with 50 nM cAMP as before (low cAMP), and the third treated with 500 µM cAMP (high cAMP) every hour for the duration of the experiment. Samples were analysed by western blot as described above.</p

CycC-CTAP protein levels in response H₂O₂ treatment.

<p>Ax2[<i>cycC</i>::<i>cycC</i>-CTAP] and <i>Ax2</i>[<i>act15::</i>FLAG<i>-cycC</i>] cells were harvested, washed and shaken in KK₂ buffer at 1×10⁷ cells/ml. After 1 hr the cells were split and incubated in the presence (+) or absence (−) of 0.25 mM H₂O₂. Western samples were taken after 1, 2 and 3 hrs of treatment and analysed on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an α-mouse IgG secondary antibody and an α-actin antibody to control for loading.</p

Comparison of cyclin C coding sequences.

<p><b>A</b>. The sequence of the predicted <i>Dictyostelium</i> protein (DDB_G0274139 at dictybase.org) was aligned against <i>Homo sapiens</i> (<i>H.s</i>.) Cyclin C and Srb11 from <i>S. pombe</i> (<i>S.p</i>.) and <i>S. cerevisiae</i> (S.c.). Alignment against a generic cyclin box from the NCBI conserved domain database (cd00043) is also shown. <b>B</b>. Cyclin protein sequences were obtained from NCBI (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) nucleotide databases and aligned using Clustal W in Bioedit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010543#pone.0010543-Chenna1" target="_blank">[36]</a>. A generic cyclin box sequence from the NCBI conserved domain database (cd00043) was used to define the cyclin box domain within each protein. These domains were used to construct a phylogenetic tree in Bioedit using the neighbour joining method. The tree was rooted using CycJ18 from <i>Arabidopsis thaliana</i> as an outgroup.</p

Analysis of myc-Cdk8 complexes in the presence and absence of FLAG-CycC.

<p><b>A</b>. Nuclear extracts from 1×10⁹ Ax2[<i>myc-cdk8</i>, FLAG<i>-cycC</i>] or Ax2[<i>myc-cdk8</i>] cells (annotated as FLAG-CycC + and - respectively) were fractionated by gel filtration on a Superose 6 column and analysed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010543#pone-0010543-g005" target="_blank">Figure 5</a>. <b>B and C</b>. Samples were harvested from vegetatively growing cells and analysed by western (B and C) and northern (C) blot. Western blots samples were analysed and probed with the α-FLAG antibody to recognise FLAG-CycC and against the myc epitope to recognise myc-Cdk8. RNA samples were resolved on a 1% formaldehyde gel, transferred to a nylon membrane and probed with a ³²P labelled fragment of the <i>cycC</i> gene. The blot was reprobed with a ³²P labelled fragment of the <i>IG7</i> gene to control for loading.</p

Signals regulating expression of cyclin C during development.

<p><b>A</b>. <b>I</b>. Exponentially growing <i>carC^-</i>[<i>cycC::cycC-</i>CTAP] and <i>gskA^-</i>[<i>cycC::cycC-</i>CTAP] cells were harvested and developed on filters at a density of 3×10⁶ cells/cm². Samples were collected at the times indicated and analysed by western blot as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010543#pone-0010543-g001" target="_blank">Figure 1A</a>. <b>II</b>. Exponentially growing <i>carC^-</i>[<i>cycC::cycC-</i>CTAP] and <i>gskA^-</i>[<i>cycC::cycC-</i>CTAP] cells were harvested and shaken in KK₂ buffer at 5×10⁶ cells/ml and pulsed with 50 nM cAMP every 5 mins. After 4 hrs cells were split and shaken in the presence (+) or absence (−) of 500 µM cAMP, added every hour for the duration of the experiment. Samples were taken at the time points indicated and analysed by western blot as above. <b>B</b>. Exponentially growing Ax2[<i>cycC</i>::<i>cycC-</i>CTAP] cells were harvested and shaken in KK₂ buffer at 5×10⁶ cells/ml, pulsed with 50 nM cAMP every 5 mins. After 4 hrs the cells were split and incubated in the presence (+) or absence (−) of 5 mM 8Br-cAMP. Samples were collected at the times indicated and analysed by western blot as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010543#pone-0010543-g001" target="_blank">Figure 1A</a>.</p

(A) Effect of PP2 on PKCδ tyrosine phosphorylation.

<p>Human washed platelets were treated with 0.1 or 1 U/ml thrombin for 1 min in the presence or absence of PP2 (10 µM, 5 min). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (B) Effect of PP2 on pleckstrin phosphorylation in thrombin-stimulated platelets. Washed human platelets were labelled with radioactive ³²P-orthophosphate and stimulated with 1 U/ml thrombin for 1 min. PP2 (10 µM, 5 min) or Ro31-8220 (10 µM, 1 min) were added where indicated. After separation by SDS-PAGE, the resulting gel was analysed using a phosphoimager to obtain radioactivity levels. Histogram shows the level of pleckstrin phosphorylation compared to basal levels. Data from one experiment, representative of two. (C) Tyrosine phosphorylation of PKCδ in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin in the absence or presence of Ro31-8220 (10 µM, 1 min) (left), or with 1 U/ml thrombin for the times shown (right) in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (D) Tyrosine phosphorylation of PKCε in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin for 1 min. Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. All studies described in this figure were performed in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Histograms show mean density of bands, normalised according to the basal value: *signifies values significant from basal, ^#signifies significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

Effect of Ro 31-8220 (Ro) on PKCδ tyrosine phosphorylation.

<p>Human washed platelets in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM) were treated with (A) 1 U/ml thrombin or (B) 3 µg/ml convulxin in the presence or absence of Ro 31-8220 (10 µM, 1 min) for the times shown. Immunoprecipitations and reprobes were carried out as in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histograms show mean density of bands, normalised according to the basal value. *signifies statistically significant from basal, ^#signifies statistically significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

Platelet function in PKCε-null mice.

<p>(A) Aggregation and ATP secretion. Washed platelets from PKCε-deficient (PKCε^−/−) mice or wild-type (WT) littermate controls were stimulated with the indicated concentrations of CRP, collagen or PAR4 peptide. Aggregation and dense granule secretion were investigated using light-scattering aggregometry and luciferin-luciferase luminescence, respectively. Traces are representative of between 3–7 mice. (B) Spreading. PKCε^−/−and wild-type (WT) washed platelets in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM) were exposed to surfaces of 100 µg/ml fibrinogen (FG), 1 U/ml thrombin (Thr), or 100 µg/ml collagen (COLL) for 45 min before fixing and mounting. Representative images from each condition are shown. Histograms depict levels of platelet adhesion to each surface and platelet mean surface area (spreading). The results are from one experiment that is representative of three. (C) Expression levels of surface glycoproteins. PKCε^−/− and WT washed platelets were incubated with FITC-labelled antisera against (upper panels) GPVI and integrin α_IIbβ₃ and (lower panels) integrin α₂β₁ and expression levels analysed by flow cytometry. The shaded area represents the signal from non-specific IgG control.</p

Presence of novel PKC isoforms in human and mouse platelets.

<p>Washed human (Hu) or mouse (Mo) platelets were lysed and samples separated by SDS-PAGE. The resulting membranes were blotted with antibodies raised against each isoform. Alongside each sample, the positive control (+) was run, as recommended by the antibody manufacturer. The results are representative of three experiments.</p