Multiple Host Kinases Contribute to Akt Activation during Salmonella Infection

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3–5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3–5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.


Introduction
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of food poisoning worldwide and infections can be lethal in young children or immunocompromised hosts [1,2]. These bacteria have the ability to invade host cells and grow intracellularly. To accomplish this, S. Typhimurium uses type 3 secretion systems (T3SS) to translocate effector proteins directly into host cells [3]. The T3SS effectors activate signal transduction pathways leading to actin rearrangements that drive internalization of the bacteria into membrane-bound compartments, known as Salmonella-containing vacuoles (SCVs), within which the bacteria can survive and replicate [4,5]. One T3SS effector, SopB, is known to contribute to S. Typhimurium invasion [6]. SopB also contributes to other important phenotypes associated with infection, including production of proinflammatory cytokines [7], controlling SCV maturation [8], intracellular positioning of the SCV [9], blocking apoptosis [10], intracellular bacterial replication [8] and induction of epithelial-mesenchymal transition [11].
SopB is an important modulator of signal transduction pathways in host cells during infection and, together with two other T3SS effectors -SopE and SopE2-SopB influences members of the small Rho GTPase family [6]. It was demonstrated that SopB activates RhoG via its guanine nucleotide exchange factor SGEF, leading to actin rearrangements that promote bacterial invasion [7]. In addition, SopB can activate the pro-survival kinase Akt, preventing apoptosis in infected cells [10,12]. SopB was found to be solely responsible for Akt activation and subsequent inhibition of caspase-3 activity, protecting infected epithelial cells from apoptosis [10]. Thus, SopB acts as a pro-survival factor, preventing apoptosis and allowing the generation of a replicative niche in infected cells. Yet, the mechanisms by which SopB promotes Akt activation are unclear.
SopB contains a catalytic domain with phosphoinositide phosphatase activity that is essential for all of its associated phenotypes [6,13,14]. It has broad substrate specificity in vitro, but in vivo substrates have been the subject of controversy and the mechanisms by which it acts are unclear. The main substrate of SopB during infection is PI(4,5) P 2 , leading to the generation of PI(5) P [15,16]. Paradoxically, despite the fact that in vitro SopB can dephosphorylate PI(3,4) P 2 and PI(3-5) P 3 , Mallo et al. showed that SopB is actually required and responsible for the accumulation of these lipids at invasion ruffles [15]. In the canonical pathway of Akt activation, e.g. during growth factor stimulation, Class I PI3-kinase generates PI(3,4) P 2 and PI(3-5) P 3 in the plasma membrane, where Akt binds and then becomes phosphorylated on Thr308 and Ser473 by PDK1 and mTORC2 complex, respectively [17][18][19]. However, the equivalent PI-accumulation during Salmonella invasion was found to be resistant to the PI3-kinase inhibitor LY294002 [15], suggesting that in this instance PI(3,4) P 2 and PI(3-5) P 3 are generated by a Class I PI3-kinase independent mechanism. Indeed, siRNA-mediated depletion of Class I PI3kinase regulatory subunits p85α and p85β had no inhibitory effect on SopB-mediated Akt activation [20]. Despite this, Steele-Mortimer and colleagues showed that PDK1 and mTORC2 are required for SopB-mediated Akt activation [20]. These authors concluded that SopB activates Akt by a mechanism independent of Class I PI3-kinase that nevertheless incorporates some elements of the canonical pathway. Still, the role of PI(3,4) P 2 and PI(3-5) P 3 in Akt activation by SopB has not been tested directly.
The mechanisms that regulate Akt activation during Salmonella infection remain unclear. Here, we show that localized PI(3,4) P 2 and PI(3-5) P 3 production at invasion ruffles is responsible for Akt activation. Furthermore, we show that SopB exploits several different host kinases, depending on the infected cell type, to activate Akt during invasion. Our studies provide important novel insight into bacterial modulation of host kinase activity that can lead to the establishment of an intracellular replicative niche.

Antibodies and Reagents
Rabbit polyclonal antibodies to Ser473-phosphorylated-Akt and pan-Akt were purchased from Cell Signaling. Vps34 antibody was described elsewhere [25] and provided by J. Backer (Albert Einstein College of Medicine). Rabbit polyclonal antibody to S. Typhimurium O antiserum Group B was from Difco. Murine monoclonal anti-GFP antibody was obtained from Invitrogen. Mouse anti-GAPDH antibody was from Milipore. Secondary antibodies used for immunofluorescence study and Phalloidin were Alexa-conjugated and purchased from Molecular Probes. Recombinant human EGF and Wortmannin were purchased from Invitrogen. LY294002 was from Cell Signaling.
S. Typhimurium infections were carried out as described previously [28]. Briefly, bacteria were grown overnight shaking at 37°C and then subcultured in LB without antibiotics for 3 hours. Bacterial inoculum was prepared by pelleting 1 mL of the subculture at 10,000 x g for 2 min, washed once and diluted 1:100 in PBS. PI3-kinase inhibitors were added 30 min prior to the infection and maintained throughout the experiment.

siRNA/esiRNA oligonucleotides and transfections
Control siRNA and those directed against Class II PI3kinases and IPMK were obtained from Sigma. Vps34 siRNA was described elsewhere [15]. esiRNA against all kinases were obtained from the SIDNET facility at the Hospital for Sick Children. Cells grown in 24-well plates were transfected with 200 nM of individual siRNAs or 200 ng of individual esiRNAs using Lipofectamine® RNAiMAX transfection reagent (Life Technologies) and used for experiments 48 hours after transfection. To confirm knockdown, RNA was isolated with an RNeasy Mini kit (Qiagen) and cDNA was transcribed with the iScript cDNA synthesis kit (BioRad). A qPCR reaction was performed using the SSO advanced SYBR green mix (BioRad).

Immunofluorescence and Western Blot analysis
Fixed cells were immunostained as described [29]. To differentiate between intra-and extracellular bacteria, cells were immunostained before permeabilization with saponin. Images were acquired with a Quorum spinning disk microscope with a 63 x oil immersion objective (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera, spinning disk confocal scan head). Quantification of samples was done with an epifluorescence microscope (DMIRE2; Leica) equipped with a 100x/NA 1.4 oil objective (Plan Apochromat; Leica).
For Western Blot analysis, cells were washed three times with PBS and then lysed with RIPA buffer. Samples were separated on 10% SDS-PAGE gels, transferred to PVDFmembranes and blocked in 5% milk for 1 hour. Primary and Secondary antibodies were incubated in 2% BSA solution. Bands were detected using the ECL detection system (GE Healthcare).

Calculation of the relative phospho-Akt expression level
Intensity of the phospho-Akt protein band on a Western blot was estimated using ImageJ software. In order to account for potential differences in the total amount of proteins from one sample to another, intensity of the phospho-Akt band was divided by that of the total Akt band. This quotient is the relative phospho-Akt expression level. In an experiment, all relative p-Akt expression levels were normalized to that of the control sample.

In vitro dephosphorylation assay and thin layer chromatography
His-SopB and His-SopB-C460S were recombinantly expressed in E. coli BL21 and affinity purified with HisTrap Ni-Sepharose columns according to Manufacturer's instructions (GE Healthcare). BODIPY® FL PI(4,5) P 2 C6 and BODIPY® FL PI(3-5) P 3 C6 were from Cedarlane. To make liposomes, the required amount of stock lipids (in chloroform) was dried under N 2 and then resuspended in 50mM Tris buffer. Liposomes were incubated with recombinant SopB and then separated on 250 µM silica gel TLC plates (Whatman) with a resolving solution of 2-Propanol, NH 4 OH and H 2 O (65:20:15). Lipids were visualized with a Storm 840 chemiluminescence imager system (Molecular Dynamics).

SopB-mediated Akt activation at Salmonella invasion ruffles is PI (3,4) P 2 /PI (3-5) P 3 -dependent
SopB is necessary for the activation of Akt during Salmonella invasion, as judged by western blotting with phospho-specific antibodies that recognize the active form of Akt, which is phosphorylated at Serine 473 (pAkt-Ser473) [12]. Cooper et al. previously showed that phosphorylated Akt is recruited to Salmonella ruffles in SopB-dependent manner [20]. Consistent with this earlier finding, we observed accumulation of pAkt-Ser473 at the actin-rich bacterial invasion site, which was visualized by phalloidin staining ( Figure 1A). Such accumulation of pAkt-Ser473 at invasion sites was not observed following infection by a bacterial SopB-deficient (ΔsopB) mutant ( Figure 1A). We also observed that cells transfected with plasmids expressing SopB showed significant increase in Akt activation compared to control (GFP-transfected) cells ( Figure S1). Thus, SopB is both necessary and sufficient for Akt activation.
We previously showed that SopB leads to the formation of PI(3,4) P 2 and PI(3-5) P 3 at bacterial invasion sites [15]. Visualization of PI(3,4) P 2 /PI(3-5) P 3 is accomplished by expression of GFP-PH-Akt, a fluorescent probe for these phosphoinositides. Wild type bacteria led to recruitment of GFP-PH-Akt to invasion ruffles, while a bacterial ΔsopB mutant did not, consistent with our previous studies [15] ( Figure 1B). Since formation of PI(3,4) P 2 and PI(3-5) P 3 at membranes is required for canonical Akt activation, we tested if these phosphoinositides are required for SopB-dependent Akt activation by inhibiting their accumulation at invasion sites. This was accomplished by expression of PTEN, a PI-3phosphatase that specifically dephosphorylates PI(3,4) P 2 and PI(3-5) P 3 . Expression of YFP-tagged PTEN-A4 (with 4 alanine substitutions S380A, T382A, T383A, and S385A) has been successfully used to block accumulation of these PIs [23]. Mutations in this construct are at the regulatory phosphorylation sites at the C-terminus of PTEN, making the resultant protein more active by targeting it to the plasma membrane [23]. After transfection of this modified PTEN construct, SopB-mediated Akt activation was almost completely abolished at invasion sites, as determined by immunofluorescence ( Figure 1C, D). A similar decrease in Akt activation can be seen by immunoblotting ( Figure 1E). Together, these results suggest that PI(3,4) P 2 /PI(3-5) P 3 production is required for Akt activation by SopB.

SopB-Mediated Akt Activation in HeLa Cells Is Only Partially Sensitive to PI3-Kinase Inhibitors LY294002 and Wortmannin
PI3-kinases, which phosphorylate PIs at the 3-position of the inositol ring, can be divided into three main classes depending on their substrate specificity [30]. We tested all 3 classes of PI3-kinases for their ability to influence SopB-mediated Akt activation. Class I PI3-kinases can be effectively inhibited by the PI3-kinase inhibitors LY294002 and Wortmannin. HeLa cells were treated with varying concentrations of LY294002 ( Figure 2) or Wortmannin ( Figure S2), then infected with wild type Salmonella. Akt activation was measured in cell lysates with anti-pAkt-Ser473 antibodies. Addition of low concentrations of LY294002 did not significantly impair SopBmediated Akt activation during infection. However, higher concentrations, e.g. 50-100 µM, LY294002 significantly reduced the Akt activation by approximately 25% compared to the control. Wortmannin showed a very similar impact on Akt activation. In contrast, Akt activation by EGF was abolished by low concentrations of either inhibitor. This indicates that Class I PI3-kinases, which are normally inhibited by LY294002 or Wortmannin, play a minor role in the SopB-mediated Akt activation in HeLa cells.

Class II PI3-Kinase beta isoform (PI3K-C2β) contributes to SopB-mediated Akt activation
Class II PI3-kinases are less sensitive to PI3-kinase inhibitors [31] and Class II-mediated Akt activation has been shown to be required for glucose-stimulated insulin secretion  [32]. Therefore, we targeted expression of all Class II PI3kinase isoforms using siRNA and analyzed Akt activation by Salmonella. Knockdown of PI3K-C2β led to a significant decrease in SopB-dependent Akt activation, while that of other Class II PI3-kinase isoforms was without effect (Figure 3). Though silencing PI3K-C2β reduced Akt phosphorylation, this kinase seemed to only have a partial role in Akt activation in HeLa cells, indicating that other kinases are also involved.

Class III PI3-Kinase Vps34 does not play a role in SopBmediated Akt activation
Class III PI3-kinase Vps34 mediates phosphorylation of PI, generating PI3P. Previous studies suggested that phosphorylation of PI3P can lead to production of PI(3,4) P 2 and PI(3-5) P 3 [33]. Therefore, we tested the role of Vps34 in Salmonella-induced Akt activation, targeting its expression with siRNA. Knockdown of Vps34 did not impair SopB-dependent activation of Akt, indicating that the Class III PI3-kinase does not contribute to SopB-mediated Akt activation (Figure 4).

SopB does not directly generate PI (3-5) P 3 by phosphotransfer
Since the canonical PI3-kinases cannot fully account for Akt activation during Salmonella invasion, we considered other possible mechanisms by which SopB could lead to the production of PI(3,4) P 2 and PI(3-5) P 3 . Because some bacterial proteins can catalyze phosphotransfer reactions between proteins [34], we considered the possibility that SopB might be mediating phosphotransfer reactions between phosphoinositides. Such a catalytic activity for SopB has not been formally tested in vitro. To determine if SopB has unrecognized catalytic activity and is by itself sufficient to induce PI(3-5) P 3 formation through a phosphotransfer reaction, we performed an in vitro assay with recombinant SopB and fluorescently tagged PIs. In our assays, SopB induced a robust dephosphorylation of PI(4,5) P 2 and PI(3-5) P 3 , generating phosphatidylinositol monophosphates ( Figure  S3). However, formation of PI(3-5) P 3 by SopB was not detected in our assay. Our studies therefore indicate that SopB does not directly mediate production of PI(3-5) P 3 during infection, but rather modulates unknown host factors to generate this phosphoinositide by a non-canonical pathway.

A kinase screen reveals that IPMK plays a role in SopBmediated Akt activation in HeLa cells
In search for the non-canonical host kinase(s) involved in SopB-mediated Akt activation, we performed an esiRNA screen of 778 mammalian kinases. The results of our screen are shown in Table 1. Many of the kinases identified have been previously implicated in regulating Akt activity (highlighted in yellow; see references), supporting the validity of our screen. The top 'hit' in our screen was inositol polyphosphate multikinase (IPMK). SopB-mediated Akt activation was impaired by as much as 91% when IPMK expression was targeted by esiRNA (Table 1). We also targeted expression of IPMK using siRNA for comparison. siRNA-mediated silencing of IPMK expression similarly impaired Akt activation, providing additional evidence for its role in HeLa cells ( Figure 5A).
To further explore the role of IPMK in SopB-mediated Akt activation, we used two different cell types lacking IPMK. In IPMK knockout embryonic stem (ES) cells [27], we observed a significant decrease in Akt activation upon Salmonella infection, compared to wild type control cells ( Figure 5B and C). We also examined mouse embryonic fibroblast cells (MEFs) lacking IPMK [35]. In contrast to ES cells, we did not see a decrease in Akt activation in IPMK -/-MEFs when infected with wild type Salmonella compared to WT MEFs ( Figure S4). However, when we treated the ES and MEF cells with the PI3-  kinase inhibitor LY294002, we saw a significant decrease in SopB-mediated Akt activation, suggesting that Class I PI3kinase may play a dominant role in these cell types unlike in HeLa cells. Together, our studies demonstrate that IPMK contributes to Akt activation in HeLa cells, but has a minor or negligible role in other tested cell types.

Discussion
Recently, we showed that PI(3,4) P 2 and PI(3-5) P 3 are generated at invasion ruffles in a SopB-dependent manner [15]. In our studies of HeLa cells, formation of these phosphoinositides and the phosphorylation of Akt were found to be sensitive to PI3-kinase inhibitors LY294002 and  wortmannin at only high concentrations, suggesting that kinases other than Class I PI3-kinase are also involved. In contrast, Cooper et al. previously showed that SopB-mediated Akt activation is resistant to 100 nM wortmannin, but completely inhibited by 50 µM LY294002 [20]. However, we believe that such differences in sensitivity may be cell-type dependent, since we saw a significant decrease in SopBmediated Akt activation when Salmonella-infected embryonic stem cells or mouse embryonic fibroblasts were treated with LY294002. Our findings and that of Cooper et al. suggest that the mechanisms of Akt activation by Salmonella vary depending on the cell types used for infection. When Class II PI3-kinase C2β was knocked down in HeLa cells, we observed that SopB-mediated Akt activation was significantly decreased, although this kinase seemed to only play a minor role. No further reduction in Akt activation was noted when such silenced cells were additionally treated with LY294002. We concluded that other kinases must be activated downstream of SopB to induce PI(3,4) P 2 /PI(3-5) P 3 formation at membrane ruffles. After performing a screen of ~780 host kinases for their potential role in SopB-mediated Akt activation, we identified about 90 kinases that influenced Akt activation after Salmonella invasion. This remarkably large repertoire of kinases that contribute to SopB-mediated Akt activation is not surprising, given that SopB is known to be able to manipulate many host kinase phosphorylation events in infected cells [36]. In fact, Rogers et al. have proposed that approximately half of all Salmonella-induced phosphorylation events in the host cell require the SopB effector [36]. Manipulation of the phosphorylation status of host kinases may be necessary for SopB-mediated PI(3,4) P 2 and/or PI(3-5) P 3 synthesis. It is noteworthy that a complete screen for kinases required for Akt activation has not been previously published. Therefore, the results of our kinase screen are likely to open up new avenues of study into the regulation of Akt.
The kinase that showed the greatest effect on SopBmediated Akt activation in HeLa cells was IPMK. IPMK is reported to have PI3-kinase activity and it was shown to phosphorylate PI(4,5) P 2 to generate PI(3-5) P 3 [37]. Also, IPMK was shown to activate Akt [26]. However, as measured by RT-PCR, IPMK knockdown efficiency was not consistent in HeLa cells, which led to variable extent of reduction in phospho-Akt expression. Thus, we decided to try 2 different knockout cell lines lacking IPMK. To our surprise, we did not see a large decrease in SopB-induced Akt activation in these cells. However, we noted a significant effect of the PI3-kinase inhibitor LY294002 on SopB-induced Akt activation in embryonic stem cells and mouse embryonic fibroblasts, suggesting that Class I PI3-kinases play a more prominent role in these cells. This suggests that SopB can recruit and/or activate various kinases in different cell types to induce PI(3,4) P 2 /PI(3-5) P 3 formation at invasion ruffles.
How SopB is producing these PIs and exploiting the host kinases needs to be further elucidated. The main substrate of SopB is PI(4,5) P 2 , generating the rare lipid PI(5) P. Since SopB does not seem to have enzymatic activity other than PIphosphatase activity, the mechanism behind SopB-mediated Akt activation might be via PI(5) P. It is conceivable that PI(5) P recruits distinct lipid kinases to Salmonella invasion ruffles to synthesize PI(3,4) P 2 and/or PI(3-5) P 3 and/or kinases that directly activate Akt independently of the lipids. In addition, PI(5) P may have an inhibitory effect on protein phosphatases that deactivate Akt, such as PP2A, or on lipid phosphatases that dephosphorylate PI(3-5) P 3 , like SHIP2.
Shigella flexneri utilizes a type 3 secreted effector IpgD, a SopB homologue, for infection. Pendaries et al. recently suggested that PI(5) P production by IpgD is necessary for activation of Class I PI3-kinases, thereby activating Akt, in HeLa cells [38]. Although both IpgD and SopB lead to the accumulation of PI(5) P, whether or not SopB-mediated Akt activation is similar to that of IpgD is unclear, because the former is only partially sensitive to PI3-kinase inhibitors in the HeLa cells. It has been shown in vitro that these two homologues have different substrate specificities [14,39]. Hence, a slight difference in the underlying mechanisms of these two bacterial effectors may be possible.
The advantage of bacterial exploitation of several host kinases instead of depending on a single kinase is obvious. Since Akt has been previously shown to play an important role in the Salmonella life cycle in the infected host cell, it would be beneficial for the pathogen to manipulate multiple kinases to ensure its activation. Additionally, our data suggests that Salmonella may have evolved to use different repertoires of kinases for Akt activation in different types of host cells. This may be intended to adapt to variable expression levels of the kinases in different cell types.
In summary, our data indicate that Salmonella can exploit different host kinases during invasion to optimize replication Cells were infected with wild type S. Typhimurium for 30 min. As a control, cells were uninfected. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (C) Western blot results of ES10 and ES5 from B were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown. doi: 10.1371/journal.pone.0071015.g005 and survival in host cells. SopB generates PI(3,4) P 2 /PI(3-5) P 3 at invasion sites via a specific group of kinases, which depends on the type of infected host cell. This PI-formation at membrane ruffles is responsible for activating Akt and inhibiting apoptosis in infected cells, as well as modulating SCV biogenesis. However, the precise mechanism of how SopB manipulates different kinases for Akt activation remains incompletely understood and needs further investigation. Figure S1. SopB expression is sufficient to activate Akt. HeLa cells were transiently transfected with plasmids encoding untagged SopB, GFP-SopB or GFP. As a control, cells were stimulated with 100 ng/mL EGF for 5 min. Akt activation of cells was assessed 8 h after transfection by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (TIF) Figure S2. SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor Wortmannin. HeLa cells were treated with different concentrations of Wortmannin (10 nM to 1000 nM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with ΔSopB mutant S. Typhimurium for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (TIF) Figure S3. SopB is not sufficient to produce formation of PI(3-5) P 3 in vitro. Reaction mixtures of FL-PIP 2 or FL-PIP 3 and His-tagged recombinant SopB or catalytically inactive SopB (C460S) were separated on TLC. As a control, pure lipids were directly separated on TLC and liposomes without recombinant SopB were also examined. (TIF) Figure S4. IPMK does not contribute to SopB-mediated Akt activation in mouse embryonic fibroblasts (MEF). (A) Wild type or IPMK knockout mouse embryonic fibroblasts were infected with wild type or ΔsopB mutant S. Typhimurium for 30 min. As controls, cells were uninfected or treated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown. (TIF)