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The Journal of General Virology logoLink to The Journal of General Virology
. 2010 Apr;91(Pt 4):858–866. doi: 10.1099/vir.0.015719-0

Herpes simplex virus type 1 latency-associated transcript inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells following serum starvation by maintaining protein kinase B (AKT) levels

Sumin Li 1, Dale Carpenter 2, Chinhui Hsiang 2, Steven L Wechsler 2,3,4, Clinton Jones 1
PMCID: PMC2888161  PMID: 19955563

Abstract

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is expressed abundantly in latently infected sensory neurons. LAT-deletion-mutant virus strains have reduced-reactivation phenotypes in small animal models of infection, demonstrating that LAT plays an important role in the latency–reactivation cycle of HSV-1. Previous studies demonstrated that the anti-apoptosis functions of LAT are important for regulating the latency–reactivation cycle because three different anti-apoptosis genes can substitute for LAT. Although LAT inhibits caspase 3 activation, the signalling pathway by which LAT inhibits caspase 3 activation was not identified. In this study, we analysed mouse neuroblastoma cells (C1300) that express LAT stably (DC-LAT6 cells) following serum starvation. As expected, DC-LAT6 cells were resistant to apoptosis following serum withdrawal. Levels of total and phosphorylated AKT (protein kinase B), a serine/threonine protein kinase that promotes cell survival, were higher in DC-LAT6 cells after serum withdrawal than in C1300 cells or a cell line stably transfected with a LAT promoter mutant (DC-ΔLAT311). A specific AKT inhibitor reduced the anti-apoptosis functions of LAT and phosphorylated AKT levels. After serum withdrawal, more DC-LAT6 cells sprouted neurites and exhibited a differentiated morphology. NeuN (neuronal nuclei), a neuron-specific nuclear protein, was expressed abundantly in DC-LAT6 cells, but not C1300 cells, after serum withdrawal, further supporting the concept that LAT enhanced neuronal-like morphology. Collectively, these studies suggested that LAT, directly or indirectly, maintained total and phosphorylated AKT levels, which correlated with increased cell survival and mature neuronal-like morphology.

INTRODUCTION

In the USA, >50 % of adults are infected with herpes simplex virus type 1 (HSV-1) (reviewed by Jones, 1998, 2003). HSV-1 is the leading cause of infectious corneal blindness in the USA, primarily because of recurrent infections. Following lytic infection in mucosal tissue, HSV-1 establishes a lifelong latency in sensory neurons. The primary site of latency is trigeminal ganglia (TG) if infection is initiated in the ocular cavity, nasal cavity or facial area.

The latency-associated transcript (LAT) is abundantly transcribed in latently infected neurons of mice, rabbits and humans (reviewed by Jones, 1998, 2003). The primary 8.3 kb LAT (Deatly et al., 1988; Rock et al., 1987; Zwaagstra et al., 1990) yields an abundant poly(A) and uncapped stable 2 kb LAT intron (Farrell et al., 1991; Krummenacher et al., 1997). LAT is detected predominantly in the nucleus, but can also be detected in the cytoplasm (Ahmed & Fraser, 2001; Nicosia et al., 1994; Thomas et al., 2002).

In mice, LAT enhances the reactivation phenotype (Leib et al., 1989). However, it is not clear whether LAT exerts its main influence on establishment, maintenance or reactivation of latency. The HSV-1 McKrae strain is frequently shed in tears of infected rabbits due to spontaneous reactivation (Perng et al., 1994). In rabbits, LAT is important for high, wild-type (wt) levels of in vivo spontaneous (Perng et al., 1994) and induced (Hill et al., 1990) reactivation from latency. LAT inhibits apoptosis in transiently transfected cells and TG of infected rabbits and mice (Ahmed et al., 2002; Inman et al., 2001; Kang et al., 2003; Perng et al., 2000) and can inhibit caspase 3 activation (Carpenter et al., 2007). Consequently, LAT can inhibit caspase 8- or caspase 9-induced apoptosis (Henderson et al., 2002; Jin et al., 2003; Peng et al., 2004), the two major apoptotic pathways (Krueger et al., 2001; Schmitz et al., 2000; Wang, 2001). The anti-apoptosis functions of LAT correlate with promoting spontaneous reactivation (Inman et al., 2001; Jin et al., 2003) and appear to be the crucial function of LAT because three different anti-apoptosis genes restore wt levels of spontaneous reactivation to a LAT null mutant virus (Jin et al., 2005, 2008; Mott et al., 2003; Perng et al., 2002b). LAT also represses productive viral gene expression in TG of mice during acute infection (Chen et al., 1997b; Garber et al., 1997).

Removal of serum from certain neuroblastoma cells induces a low percentage of these cells to sprout neurites and develop a differentiated morphology (Schubert et al., 1969; Seeds et al., 1970). Certain growth factors also stimulate neurite sprouting (Evangelopoulos et al., 2005; Lee et al., 2006). Neuron survival is promoted by the AKT protein kinase (Das et al., 2007), a serine/threonine protein kinase (reviewed by Manning & Cantley, 2007). Active AKT is phosphorylated at threonine 308 and serine 473 (Scheid & Woodgett, 2003). AKT can phosphorylate and regulate several pro- or anti-apoptotic proteins (reviewed by Cooray, 2004), supporting a role for AKT during apoptosis and neuronal differentiation.

In this study, we demonstrate that, following serum withdrawal, C1300 mouse neuroblastoma cells expressing high levels of LAT are more resistant to cell death than the parental C1300 cells or C1300 cells that contain a LAT promoter deletion mutant. Furthermore, more C1300 cells expressing LAT exhibit neuronal-like morphology following serum withdrawal. LAT coding sequences also maintained total AKT levels and phosphorylated AKT at serine 473. A specific inhibitor of AKT reduced survival of DC-LAT6 cells after serum starvation, suggesting that active AKT is important for the anti-apoptosis functions of LAT.

RESULTS

LAT decreases serum-starvation-induced cell death

To test whether LAT (Fig. 1a, b) has anti-apoptosis activity in C1300 cells following serum depletion, cells were cultured in serum-free medium and cell survival was quantified. For these studies, C1300 cells stably transfected with a NotI–NotI LAT fragment were used (Fig. 1c; DC-LAT6). DC-LAT6 cells express high levels of LAT and are resistant to cold-shock-induced apoptosis (Carpenter et al., 2007). DC-ΔLAT311 contain a NotI–NotI fragment lacking the LAT promoter and these cells are not resistant to cold-shock-induced apoptosis (see Fig. 1d for schematic showing the fragment used to generate the DC-ΔLAT311 cell line). The percentage of surviving cells 7 days after serum starvation, as judged by trypan blue exclusion assay of cell viability, was significantly higher for DC-LAT6 cells (Fig. 2a; column L) than for C1300 or DC-ΔLAT311 cells (columns C or Δ, respectively) (P<0.05 using Student's t-test). DC-ΔLAT311 cells also appeared to have a lower percentage of surviving cells than C1300 cells.

Fig. 1.

Fig. 1.

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Schematic of viruses used in this study and the organization of LAT. (a) Genomic structure of wt HSV-1. The HSV-1 genome contains a long unique region (UL) bounded by inverted long repeats (TRL, terminal repeat long; IRL, internal repeat long) and a short unique region (US) bounded by inverted short repeats (IRS, internal repeat short; TRS, terminal repeat short). The LAT locus is located in the long repeats and is therefore present in two copies per genome. The crossing dashed lines indicate that the expanded view of the LAT regions in (b) represents both LAT copies in opposite orientations. (b) Expanded view of LAT locus and genes within long repeats. The large arrow denotes the primary LAT transcript. The solid rectangle represents the stable 2 kb LAT intron. The LAT TATA box is indicated by TATA. The start of LAT transcription is denoted by the arrow at +1 (genomic nt 118801). Sequences from LAT nt 1 to 661 comprise exon 1. Restriction-enzyme sites and relative locations of the ICP0 and ICP34.5 transcripts are shown for reference. (c) Schematic of LAT sequences used to stably transfect C1300 mouse neuroblastoma cells. Partial restriction map of LAT sequences in the fragment used to construct DC-LAT6 cells. The NotI–NotI fragment used to construct DC-LAT6 cells was derived from strain 17syn+. (d) A PstI deletion construct was also used to stably transfect C1300 cells and these cells were designated DC-ΔLAT311 (Carpenter et al., 2007). This deletion is denoted by XXX beneath the schematic.

Fig. 2.

Fig. 2.

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LAT inhibits apoptosis following serum withdrawal. (a) DC-LAT6 (column L), DC-ΔLAT311 (column Δ) or C1300 cells (column C) (approx. 1×105 cells per 60 mm dish) were serum-starved for 7 days and the percentage of trypan blue-positive cells was determined. Values shown are the means+sem of three different experiments and each experiment was performed in triplicate. *Difference between DC-LAT6 and C1300 or DC-ΔLAT311 cells was significant (P<0.05, t-test). (b) Serum withdrawal was performed for the designated time after plating the respective cell lines. Collection of apoptotic DNA was performed as described in Methods. Apoptotic DNA was separated on a 2 % agarose gel. Levels of apoptotic DNA were measured using a Bio-Rad Molecular Imager FX and expressed as the fold apoptosis induction (day 0 was arbitrarily assigned a value of 1.0). Lane M is a 100 bp DNA molecular mass marker and the bottom band is 100 bp. Lane CS (cold-shock-induced apoptosis) served as a positive control for apoptosis and was performed as described in Methods. The results are representative of three independent experiments.

As expected, little or no apoptotic DNA was detected in the three cell lines at 0 or 1 day after serum withdrawal (Fig. 2b). As a positive control for apoptosis, C1300 cells were subjected to cold-shock-induced apoptosis (lane CS) as described previously (Carpenter et al., 2007; Shen & Jones, 2008). Cold-shock-induced apoptosis is reproducible in C1300 cells. Two days after serum removal, higher levels of apoptotic DNA were detected in C1300 cells (Fig. 2b, lane C) than in DC-LAT6 cells (lane L). Although lower levels of apoptotic DNA were detected in DC-ΔLAT311 cells on day 2 and 3 after serum withdrawal (lane Δ), more of these cells died as judged by trypan blue exclusion studies (Fig. 2a; data not shown), suggesting that these cells died by a non-apoptotic death following serum withdrawal. In summary, this study indicated that LAT coding sequences inhibited cell death following serum withdrawal, which is consistent with previous conclusions demonstrating that LAT inhibits apoptosis (Ahmed et al., 2002; Inman et al., 2001; Kang et al., 2003; Perng et al., 2000).

LAT stabilizes AKT protein levels in serum-starved DC-LAT6 cells

As the protein kinase AKT promotes cell survival and phosphorylated AKT is the active kinase (Manning & Cantley, 2007; Scheid & Woodgett, 2003), we compared AKT phosphorylation in DC-LAT6 cells with C1300 and DC-ΔLAT311 cells using an antibody that recognizes phosphorylated AKT on serine 473, which is the active form of AKT (Alessi et al., 1996). Phosphorylated AKT levels were significantly higher in DC-LAT6 cells (Fig. 3a, lane L and Fig. 3b, grey bars) than in C1300 (lane C or black bars) or DC-ΔLAT311 (lane Δ or white bars) cells after serum depletion for 7 days (P<0.05). Total AKT levels were also significantly higher (P<0.05) in DC-LAT6 cells relative to β-actin levels on days 5 and 7 after serum starvation (Fig. 3c).

Fig. 3.

Fig. 3.

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Analysis of phosphorylated AKT following serum withdrawal. (a) AKT phosphorylation was examined in DC-LAT6 (L), DC-ΔLAT311 (Δ) or C1300 (C) cells after serum withdrawal (days). Antiserum that recognizes AKT phosphorylated on serine 473 (p-AKT), total AKT or β-actin was used for these studies. Each lane was loaded with 100 μg protein of cell lysate. These results are consistent with at least five independent studies. (b, c) Ratios of p-AKT or total AKT to β-actin were measured using a Bio-Rad Molecular Imager FX. The results were derived from five independent experiments and error bars show sem. *Significant differences between DC-LAT6 (grey bars) and C1300 (black bars) or DC-ΔLAT311 (white bars) cells (P<0.05, t-test).

LAT coding sequences stimulate neurite sprouting in C1300 cells after serum starvation

To test whether LAT influenced neuronal differentiation, cells were cultured in medium lacking serum for 10 days. The DC-LAT6 cell line contained more attached cells that excluded trypan blue and contained more neurites than did C1300 cells (Fig. 4a). Approximately 28 % of DC-LAT6 cells (Fig. 4b, column L) had a differentiated morphology, whilst significantly fewer C1300 cells (8 %) (column C) were differentiated (P<0.003). Although DC-ΔLAT311 cells appeared to contain very low levels of cells with neurites, it was difficult to obtain consistent results because more cells died relative to C1300 cells (Fig. 2a).

Fig. 4.

Fig. 4.

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Analysis of neurite sprouting in DC-LAT6 cells following serum withdrawal. The respective cell lines were photographed after 10 days of serum withdrawal (a). The number of differentiated cells was quantified as previously described (Lee et al., 2006) (b). In brief, differentiated cells were defined as cells bearing neurites at least twice the diameter of the cell body. Results shown are the mean ± sem of four independent experiments, which were performed in triplicate. *, Difference between DC-LAT6 (L) and C1300 (C) cells was significant (P<0.05, t-test).

Total and phosphorylated AKT protein levels, but not β-actin, were higher in DC-LAT6 cells (lane L) than in C1300 cells (lane C) after 10 days of serum withdrawal (Fig. 5a). Prior to serum starvation, AKT levels were similar in C1300 and DC-LAT6 cells. DC-ΔLAT311 cells also appeared to exhibit a similar pattern to C1300 cells (data not shown). As more DC-ΔLAT311 cells than C1300 cells died after 10 days of serum withdrawal, it was difficult to consistently obtain sufficient quantities of proteins for these studies. Higher levels of GSK-3β, total (GSK lanes) and phosphorylated (p-GSK lanes), were detected in DC-LAT6 cells after serum withdrawal (Fig. 5b, lane L) than in C1300 cells (lane C), which was expected because the protein kinase GSK-3β is phosphorylated by AKT (Srivastava & Pandey, 1998; Welsh et al., 1996). Prior to serum starvation, GSK levels were similar in C1300 and DC-LAT6 cells, suggesting that growth factors in serum maintained total and phosphorylated AKT, as well as GSK protein levels.

Fig. 5.

Fig. 5.

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LAT maintains GSK and NeuN following serum withdrawal. (a) Cell lysate was prepared after withdrawal of serum for 10 days and Western blots performed using antibodies directed against phospho-AKT (p-AKT), total AKT or β-actin (100 μg protein in each lane). These results are representative of five independent studies. (b) Western blots were performed using antibodies directed against NeuN, phospho-GSK (p-GSK), total GSK or β-actin. Each lane was loaded with 250 μg protein. The images were representative of four different experiments. C, C1300 cells; L, DC-LAT6 cells.

NeuN, a nuclear protein expressed in most neuronal cells, is a neuronal differentiation marker whose expression correlates with neurite outgrowth following serum withdrawal (Englund et al., 2005; Mullen et al., 1992; Shuangshoti et al., 2005). Following 10 days of serum withdrawal, higher levels of NeuN were present in DC-LAT6 cells (Fig. 5b, lane L) than in C1300 cells (lane C). Conversely, β-actin protein levels were similar.

Inhibition of AKT activity reduces the ability of DC-LAT6 cells to survive serum withdrawal

To test whether AKT played a role in the anti-apoptosis functions of LAT, DC-LAT6 cells were treated with a specific inhibitor of AKT (AKT VIII) and then the percentage of surviving cells was measured after serum withdrawal. Five days after serum withdrawal, the percentage of surviving cells was reduced by approximately fourfold following treatment with AKT VIII (, [AKT VIII (+)] compared with samples treated with the solvent (Control) (Fig. 6a, b). DC-LAT6, C1300 or DC-ΔLAT311 cultures treated with AKT VIII contained many cells that were detached from the dish, suggesting that inhibiting AKT in the absence of growth factors led to cell death, not merely growth arrest. AKT VIII treatment also reduced phosphorylated AKT levels in DC-LAT6 cells (Fig. 6c), which was expected. These findings suggested that active AKT levels were important for the ability of LAT to inhibit apoptosis.

Fig. 6.

Fig. 6.

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AKT promotes cell survival in DC-LAT6 cells. DC-LAT6 cells were plated as described in Methods (2–3×105 cells were plated for each 60 mm dish). The following day medium lacking serum was added. Some cultures were treated with 0.5 μM AKT inhibitor (AKT VIII). Control cultures were treated with the solvent that was used to dissolve AKT VIII (DMSO). Cells were photographed 5 days after treatment (a; ×10) and the number of attached cells that were alive, as judged by trypan blue exclusion, were counted (b). The values are expressed as a percentage of total cells that survived and error bars represent the sem. *, Difference between the sample containing AKT VIII [AKT VIII(+)] and without AKT VIII (Control) was significant (P<0.05). (c) Western blot (100 μg protein in each lane) after 5 days treatment with AKT VIII using an antibody that recognizes total AKT, phosphorylated AKT (p-AKT) or β-actin.

DISCUSSION

In this study, evidence is provided suggesting that LAT coding sequences, directly or indirectly, maintain total AKT levels and AKT phosphorylation on serine 473 after serum withdrawal. In addition to LAT, sequences used to generate the DC-LAT6 cell line also express the AL transcript (antisense to the 5′ end of LAT and the LAT promoter) and its protein (Perng et al., 2002a) and the AL3 transcript and protein (Jaber et al., 2009). Although AL3 is expressed during latency, it does not appear to inhibit apoptosis directly, because AL3 (mRNA and protein) are expressed in DC-ΔLAT311 cells (Jaber et al., 2009). Two small non-coding RNAs within LAT were recently shown to inhibit cold-shock-induced apoptosis (Shen et al., 2009), suggesting that they may influence AKT protein levels and phosphorylation after serum withdrawal. AKT was important with respect to LAT inhibiting apoptosis and promoting neurite sprouting because the AKT inhibitor, AKT VIII, reduced DC-LAT6 cell survival. It is also possible that AKT promotes cell survival after serum withdrawal and additional signalling pathways promote neurite outgrowth. Several studies have demonstrated that LAT inhibits apoptosis (Hamza et al., 2007; Inman et al., 2001; Jin et al., , 2002, 2003, 2004; Peng et al., 2003, 2004; Perng et al., 2000), but this study was the first to link AKT to the anti-apoptosis functions encoded within LAT coding sequences.

The finding that LAT coding sequences promoted neurite outgrowth in C1300 cells is consistent with a previous finding that LAT enhances survival of sympathetic and TG neurons (Hamza et al., 2007). In both studies, the stable 2 kb LAT and the 3′ end of exon 1 were used, suggesting that at least a portion of this region promotes neurite growth. Regeneration and elongation of axons following damage promote neuronal survival (Caroni, 1997; Chen et al., 1997a; Horner & Gage, 2000), suggesting that LAT may promote axonal regeneration or neurite sprouting following viral infection of neurons.

AKT is important for several latent/persistent viruses (Cooray, 2004). For example, Epstein–Barr virus (EBV)-encoded LMP1 interacts with the p85 adaptor subunit of phosphatidylinositol 3-kinase, which stimulates phosphorylation of AKT and consequently B-cell survival is enhanced (Dawson et al., 2003). EBV reactivation from latency also requires active AKT (Darr et al., 2001). In general, these studies concluded that AKT activation correlates with survival of latently infected cells, thus enhancing establishment and/or maintenance of latency.

METHODS

Cell lines.

Mouse neuroblastoma C1300 cells and C1300 cell lines stably expressing high levels of LAT were previously described (Carpenter et al., 2007). DC-LAT6 cells were stably transfected with the plasmid containing the first 3225 nt of the primary LAT transcript. DC-ΔLAT311 cells lack the LAT TATA box and surrounding promoter sequences and do not express detectable levels of LAT (Carpenter et al., 2007). Cell lines were grown in Eagle's minimal essential medium (EMEM; Sigma) with 10 % fetal calf serum (FCS; Sigma), penicillin (10 U ml−1; Sigma) and streptomycin (100 μg ml−1; Sigma) in a 37 °C incubator with 5 % CO2.

Cold-shock-induced apoptosis and collection of apoptotic DNA.

Cold-shock-induced apoptosis was essentially performed as described previously (Geiser et al., 2008; Jin et al., 2004; Shen et al., 2009; Shen & Jones, 2008). In brief, cells were plated at a density of 1×106 cells in complete growth medium in a T25 plastic flask. Twenty-four hours later, fresh medium containing 2 % FCS was added to cultures. After 12 h incubation, cells containing 2 % FCS were incubated on ice (4 °C) for 60 min with the caps of the flask sealed using Parafilm. At the end of 1 h on ice, the caps were loosened and flasks were incubated at 37 °C for the designated time. Cells (1×106) were collected and apoptotic DNA prepared as previously described (Geiser et al., 2008; Jin et al., 2004; Shen & Jones, 2008). At the indicated times after cold-shock-induced apoptosis, adherent and non-adherent cells were collected and pelleted by low-speed centrifugation (2000 g for 10 min at 4 °C). Cell pellets were suspended in 400 μl hypotonic buffer [50 mM Tris/HCl (pH 8.0), 5 mM EDTA, 50 μg RNase ml−1 (Sigma), 2 % TX-100 (Sigma)] at 4 °C for 2 h. Nuclei were removed by centrifugation (10 000 g for 10 min at 4 °C). Fragmented DNA in the supernatant was immediately loaded on to a DNA-binding column (Sigma), centrifuged for 1 min at 12 000 g, washed with 500 μl wash solution (Sigma) and then the column was washed with 750 μl 70 % ethanol. The bound DNA was eluted by adding 75 μl TE (pH 8.0) to the column and centrifuging the column at 12 000 g for 2 min.

Apoptotic DNA from 3×105 cells was loaded on to a 2 % agarose gel. Using a Bio-Rad Molecular Imager FX, a high-resolution photograph of the ethidium bromide (EtBr)-stained gel was obtained. The intensity of the EtBr-stained DNA in each lane was measured by using a Molecular Imager FX and a raw value representing the intensity of the stained DNA was obtained. The values for the negative controls were set at 100 % and the values of other samples were normalized relative to the negative controls. When duplicate samples were examined using this protocol, we consistently observed the same levels of apoptotic DNA.

Cell survival and differentiation assay.

The designated cell lines were seeded on to six-well plates at a density of 100 000 cells per well and allowed to attach overnight (Lee et al., 2006). EMEM with 0.1 % bovine serum albumin (BSA; Sigma) and no FCS was added the next day and replaced every 2 days. The number of living cells that were attached to the plastic was quantified using a haemocytometer and trypan blue exclusion assay.

Differentiation of C1300 cells was performed as described previously (Lee et al., 2006). The respective C1300 neuroblastoma cell lines were seeded on six-well plates (20 000 cells per well) or 10 cm dishes (20 000 cells per dish) in complete medium (EMEM with 10 % FCS and antibiotics). The next day, medium was replaced with EMEM containing 0.1 % BSA without serum. Differentiation was defined as cells bearing neurites at least twice the diameter of the cell body. Images were captured using a Nikon digital camera.

The AKT inhibitor, AKT VIII (Calbiochem, catalogue no. 124018) was dissolved in DMSO. AKT VIII is a cell-permeable quinoxaline compound that selectively inhibits AKT1 and AKT2 activity (IC50=58 and 210 nM for AKT1 and AKT2, respectively, in an in vitro kinase assay). Inhibition appears to be pleckstrin homology domain-dependent, but is specific for AKT. AKT VIII has no inhibitory effect against other protein kinases, e.g. protein kinase A or protein kinase C (Barnett et al., 2005).

Western blotting and antibodies used for these studies.

Western blotting was performed as described previously (Saira et al., 2007; Workman et al., 2009). Cells were washed with PBS and suspended in NP-40 lysis buffer [100 mM Tris (pH 8.0), 1 mM EDTA, 100 mM NaCl, 1 % NP-40, 1 mM PMSF and one tablet of complete protease inhibitor (Roche Molecular Biochemicals) per 10 ml]. Cell lysate was incubated on ice for 30 min, sonicated and then clarified by centrifugation at 10 000 g at 4 °C for 15 min. Protein concentrations were quantified by Bradford assay. For SDS-PAGE, proteins were mixed with an equal amount of 1×sample loading buffer [62.5 mM Tris/HCl (pH 6.8), 2 % SDS, 50 mM dithiothreitol, 0.1 % bromophenol blue, 10 % glycerol] and boiled for 5 min. Proteins were separated in a 12 % SDS-PAGE gel. After electrophoresis, proteins were transferred onto a PVDF membrane (Immobilon-P; Millipore) and blocked for 4 h in 5 % non-fat dry milk with Tris-buffered saline/0.1 % Tween 20 (TBS-T) [1×TBS is 20 mM Tris base (pH 7.6), 140 mM NaCl]. Membranes were then incubated with primary antibody overnight at 4 °C. After 45 min washing with TBS-T, blots were incubated with the designated secondary antibody (2 h) that was diluted in 5 % non-fat milk in TBS-T. Blots were washed for 45 min with TBS-T and exposed to Amersham's ECL (enhanced chemiluminescence) reagents and then autoradiography was performed.

An anti-AKTser473 polyclonal rabbit antibody (1 : 1000 dilution) or anti-AKT rabbit polyclonal antibody (1 : 2000 dilution; Cell Signaling Technology, catalogue #9271 or #9272, respectively) was used for Western blots. Antiserum directed against phospho-GSK-3 beta (Cell Signaling Technology, catalogue #9336) is a rabbit monoclonal antibody that was used for Western blotting at a dilution of 1 : 500. The GSK-3β antibody (Cell Signaling Technology, #9315) is a rabbit monoclonal antibody that was used for Western blot assays at a 1 : 500 dilution. NeuN (Chemicon, MAB377) is a mouse monoclonal antibody that was used for Western blots at a 1 : 500 dilution. The β-actin antibody (Santa Cruz, catalogue no. SC-1616, 1 : 500 dilution) is a goat polyclonal antibody that was used to confirm that similar levels of proteins were loaded in each lane. The secondary antibody was diluted 1 : 3000 for donkey anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) (Amersham Biosciences), 1 : 2000 for donkey anti-goat HRP-conjugated IgG or 1 : 2000 for sheep anti-mouse HRP-conjugated IgG in blocking buffer. A Bio-Rad Molecular Imager FX was used to obtain a high-resolution image of the autoradiograph. The intensity of the respective bands in each lane was measured using a Molecular Imager FX and a raw value representing the intensity of the band was obtained. The values were then normalized relative to β-actin levels.

Statistical analysis.

All experiments were performed at least three times. All values are presented as mean±sem. Statistical comparisons of the mean values were performed with Student's t-test. P<0.05 was considered to indicate statistical significance.

Acknowledgments

The laboratory of C. J. is supported by an NIAID grant (R21AI069176), two USDA grants (08-00891 and 09-01653) and a grant to the Nebraska Center for Virology (1P20RR15635). The lab of S. L. W. is supported by Public Health Service grants (EY13191 and EY016663), the Discovery Eye Foundation, the Henry L. Guenther Foundation and Research to Prevent Blindness (RPB). S. L. W. is an RPB Senior Scientific Investigator.

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