α-MHC-tTA hearts were protected against I/R injury <i>in vitro</i> using the Langendorff-perfused heart.

<p>(A–B) LVDP was similar between α-MHC-tTA and control hearts at baseline; however, after 30 min of ischemia, control hearts recovered to 35% of baseline LVDP values whereas α-MHC-tTA had 90% recovery. (C–D) LV systolic and diastolic functions assessed by dP/dt_max and dP/dt_min respectively were significantly higher in α-MHC-tTA compared to control after 30 min of ischemia. (N = 3 for control and N = 4 for αMHC-tTA, p<0.05).</p

Biological activity of PI3KC2β versus Raf RBDs.

<p>(A) Expression of the Raf-RBD but not the PI3KC2β-RBD or GST alone inhibited EGF-stimulation of a Gal-Elk reporter assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045360#pone.0045360-Adams1" target="_blank">[23]</a>. GST-RBDs were expressed equally. GST alone migrated at a faster rate and was not visible in this image. Results represent the average relative activation ± S.E.M. from at least three independent experiments. (*p<0.05 compared to unstimulated GST, **p<0.01 compared to EGF stimulated GST). (B). PI3KC2β-RBD dose-dependently inhibits the effect of Ras17N on Src-mediated transformation. NIH/3T3 cells were transfected with 100 ng of SrcY527F expression construct in the presence or absence of Ras17N. Co-expression of the PI3KC2β-RBD reverses the inhibitory effect of Ras17N on Src transformation whereas the Raf-RBD does not. In contrast, expression of the Raf-RBD alone, but not the PI3KC2β-RBD, significantly inhibited Src-mediated transformation. The results represent the average relative focus forming activity ± S.E.M. from three independent experiments performed in triplicate. Asterisks denote samples that were significantly different from Src alone (*p<0.05).</p

Echocardiographic Measurements and Heart weight/tibia length.

<p>Values are mean ± SEM. <i>P</i> values are based on 1-tail Student's <i>t</i>-test assuming unequal variance.</p

Reduced cardiac muscle damage in α-MHC-tTA hearts subjected to I/R injury.

<p>Lack of cardiac muscle cell damage was apparent in α-MHC-tTA compared to control where abundant creatine kinase levels were observed within 15 min of start of reperfusion. (N = 4, p<0.05).</p

Effect of PI3K inhibition on the protection against I/R injur in α-MHC-tTA hearts.

<p>Administration of LY294002 did not abolish the protective effect seen in α-MHC-tTA hearts however it did abolish protection imparted by IPC. (N = 5 for α-MHC-tTA and N = 3 for α-MHC-tTA+8 µM LY294002, p<0.05).</p

Mutations in the effector region of Ras disrupt PI3KC2β binding.

<p>(A) Point mutations in the effector region of Ras12V disrupt interactions with specific Ras targets. (B) VC-tagged PI3KC2β was co-transfected with either VN-tagged Ras17N, 17N/69N, or one the effector mutants in the background of Ras17N/69N. BiFC signal is pseudo-colored green. Effector mutations that disrupt Class I PI3K binding to Ras12V disrupt Class II PI3K binding to Ras17N/69N. CFP (red) was used as a transfection control. (C) Graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments. (*p = 0.02). (D) Western blot analysis demonstrates equal expression of all constructs. (E) Mutation of Thr392 to Asp or Lys379 to Ala in full-length PI3KC2β disrupts interaction with Ras17N. The ΔRBD mutant was also included as a negative control. Graph represents the average of three independent experiments (*p<0.05).</p

Ras is necessary for ITSN1 activation of AKT.

<p>(A) YFP-ITSN1 overexpression stimulates AKT activation as measured by levels of phospho-AKT as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045360#pone.0045360-Das1" target="_blank">[22]</a>. Co-expression of Ras17N or Ras17N/69N inhibits this response. Results represent the average fold activation of AKT ± S.E.M. from at least three independent experiments. (B) Western blot analysis of AKT activation from a representative experiment. Top two panels represent Western blots of HA immunoprecipitates of cell lysates to assess AKT activation as described in the Materials and Methods section. The lower three panels indicate the level of expression of ITSN1, Ras, and actin (a loading control).</p

Ras forms a complex with PI3KC2β.

<p>(A) PI3KC2β preferentially interacts with Ras17N. VC-tagged PI3KC2β was co-transfected with one of the following VN-tagged Ras constructs: WT, 61L, 17N, or 17N/69N. BiFC signal (green) demonstrates that PI3KC2β interacted with Ras17N >17N/69N >WT >61L. CFP (red) was used as a transfection control. The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent (*p<0.05). Western blot analysis demonstrates equivalent expression of all constructs. (B) PI3KC2β does not interact with Ras12V. VC-tagged PI3KC2β was co-transfected with one of the following VN-tagged Ras constructs: WT,17N, or 12V. BiFC signal (green) demonstrates that PI3KC2β interacted with Ras17N >WT >12V. CFP (red) was used as a transfection control. The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05). Western blot analysis demonstrates expression of all constructs (size bar  = 20 μm).</p

Binding of PI3KC2β-RBD to Ras.

<p>(A) Nucleotide loaded Ras does not directly interact with the RBD of PI3KC2β. Ras-GDP or Ras-GTPγS were incubated with GST-Raf-RBD, GST-PI3KC2β-RBD or GST alone as a negative control. Bound proteins were then analyzed by Western blot with a Ras antibody. The RBD of Raf specifically bound Ras-GTPγS. Neither GST-PI3KC2β-RBD nor GST alone interacted with Ras-GDP or Ras-GTPγS. Top panel, Ras bound to GST proteins. Bottom panels, input amounts of proteins. (B) Nucleotide-free Ras was generated in vitro as described and then tested for binding to the various GST proteins as in (A). GST-PI3KC2β-RBD directly binds nucleotide-free Ras while little association was seen with the GST-Raf-RBD or GST alone. Panels are same as in A. (C) Repeat of (B) except nucleotide (1 mM) was present during the binding reaction. Panels are same as in (A). (D) Addition of nucleotide (1 mM) does not disrupt pre-bound PI3KC2β-RBD- nucleotide-free Ras. GST-PI3KC2β-RBD was first bound to nucleotide-free Ras. Following binding, the complex was incubated with 1 mM GDP or GTPγS at RT for 30 min and then washed with buffer. Bound proteins were then analyzed as in (A).</p

Ras, PI3KC2β, and ITSN1 co-localize on intracellular vesicles.

<p>(A) VN-Ras and VC-PI3KC2β (green) were co-transfected with CFP-ITSN1 (red) into COS cells. a. The PI3KC2β-Ras BiFC complex co-localizes with ITSN, represented by yellow in the overlay panel; b. The VN-Ras and VC-PI3KC2β YFP signal (green) does not bleed into the CFP channel; c. The CFP-ITSN1 signal does not bleed into the YFP channel (size bars  = 20 μm). Note: the differences in signal strength of the BiFC signal in (a) vs (b) are due to a lower power setting for the laser in (b) so that pixel intensities can be accurately quantified and are not saturated. In (a) a higher laser power was used to illustrate the punctate localization of the Ras-PI3KC2β complex throughout the cell. (B) Ras interaction with PI3KC2β is disrupted by deletion of the RBD (ΔRBD) but not by mutation of the Pro-rich, ITSN1 binding sites (PRD-PA). The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05, WT vs ΔRBD, PRD-PA vs ΔRBD). Western blot analysis demonstrates equal expression of all constructs (size bars  = 20 μm).</p