Effects of the indicated protein kinase inhibitors on site-specific phosphorylation determined using Western blots prepared using whole body protein extracts of <i>Drosophila</i> treated with various protein kinase inhibitors.

<p>Shown in panels A, B and C are the results obtained with antibodies specific to phosphorylated and total JNK; in panel D and E, the results obtained with antibodies to phosphorylated and total AMPK; and in panel F the results from antibodies to phosphorylated substrates of PKC. The inhibitors used in each study are indicated at the bottom of the panels. The symbols are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g002" target="_blank">Figure 2</a>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s005" target="_blank">Figure S5</a> for representative Western blots. In panel E, the control phosphorylated-AMPKα expression levels are derived from the averages of only 2 independent replicates, due to sample loss. All other data are derived from 3 independent replicates. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s005" target="_blank">Figure S5</a> for representative Western blots.</p

The level of phosphorylated AMPKα (white bars) and non-phosphorylated MPKα1+α2 (black bars) determined using Western blots prepared using protein extracts of S2 cells treated for 48 hours with the protein kinase inhibitors.

<p>Panels A and B show the data from one of two Western blots using control cells or cells treated with the indicated inhibitors. The labeling and symbols are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g002" target="_blank">Figure 2</a>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s007" target="_blank">Figure S7</a> for representative Western blots.</p

Effects of the protein kinase inhibitors on intracellular signaling in <i>Drosophila</i> S2 cells treated for 48 hours with the protein kinase inhibitors indicated at the bottom of the panels.

<p>Panel A shows data obtained using antibodies to phospho-Erk1/2 (white bars), and total Erk1/2 (black bars). Panel B shows data obtained using antibodies to phospho-Mek1/2 (white bars), and total Mek1/2 (black bars). Panel C shows data obtained using antibodies to phospho-p38 MAPK (white bars), and total p38 MAPK (black bars). Panel D shows data obtained using antibodies to phospho-JNK (white bars), and total JNK (black bars). The height of each bar represents the the signal normalized to the total amount of protein in each sample as judged by comparison to several apparently invariant protein bands observed on the blot by Ponceau S staining (representative staining is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s004" target="_blank">Figure S4A</a>). One asterisk indicates the changes were significant (P≤0.05); two asterisks indicates the results were highly significant (P≤0.01), and three indicates it is very highly significant (P≤0.001). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s004" target="_blank">Figure S4A</a> for representative Western blots and protein bands visualized using Ponceau S staining of membranes.</p

An abridged, consensus, protein-kinase signaling network assembled by examination of the <i>Drosophila</i> and mammalian literature.

<p>An abridged, consensus, protein-kinase signaling network assembled by examination of the <i>Drosophila</i> and mammalian literature.</p

Summary of protein kinase activity studies.

a<p>Down or up arrows indicate a decrease or increase in signal relative to control, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g002" target="_blank">Figure 2</a>). NC indicates no change. A “s” adjacent to an arrow indicates that the change was small in magnitude. Arrows in parenthesis indicate changes found in Western blots of protein extracts from drug treated <i>Drosophila</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g008" target="_blank">8</a>).</p

The effects of protein kinase inhibitors on the activation of ERK1/2 signaling in intact <i>Drosophila</i>.

<p>ERK1/2 activation in <i>Drosophila</i> fed the inhibitors in their food were determined using Western blots probed with a phosphorylation site- or total protein-specific antibody. The labeling and symbols are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g002" target="_blank">Figure 2</a>. Panels A through C represent data from 3 different Western blots. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s008" target="_blank">Figure S8</a> for representative Western blots.</p

Results obtained with the inhibitors which passed the initial screening.

a<p>Percentages indicate the average percent increase in mean lifespan obtained in the number of independent trials indicated in parentheses. Because maximum lifespan (lifespan of the longest lived 10%) was variable due to the presence of occasional long-lived outliers, this metric was not used.</p>b<p>NC indicates that the effects of the drugs on food consumption were not statistically different than control in FPAs and/or CAFE assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#s3" target="_blank"><i>Materials and Methods</i></a> for the details of the assays and statistical tests used). These FPAs used 6 control and 6 treated 8 ounce fly bottles containing 30 to 50 flies each. The absence of a notation indicates that the test was not performed for that drug. Where a range of optimum concentrations of the drug is given, the assay was performed at the highest concentration in the range.</p>c<p>H8 was rapamycin (sirolimus) in the Biomol library. Because of its poor bioavailability, we tested everolimus, its more bioavailable derivative.</p

PKC activity determined using antibodies to phosphorylated substrates of PKC in extracts of S2 cells treated for 48 hours with the indicated protein kinase inhibitors.

<p>Panels A and B show results for different subsets of the inhibitors. The labeling and symbols are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone-0029782-g002" target="_blank">Figure 2</a>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029782#pone.0029782.s006" target="_blank">Figure S6</a> for representative Western blots.</p

Food consumption as measured by FPA does not change in response to simvastatin or methoprene.

a<p>These data are the means plus or minus the standard error of the plaque number per cm² per fly per hour. The plaque values are not significantly different as judged by ANOVA followed by Tukey's multiple comparison test. See <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039581#s4" target="_blank">Materials and Methods</a></i> for specifics regarding the studies.</p

Locomotor activity of L744832 and GGTI-298 treated <i>Drosophila</i>.

1<p>Groups of 10 newly eclosed Drosophila were maintained for 14 days at 25°C with medium containing either an equal volume of vehicle, 20 µM L744832, or 300 µM GGTI-298 in a Drosophila Activity Monitoring System.</p