Journal: PLOS Pathogens

Article Title: Inhibition of Src signaling induces autophagic killing of Toxoplasma gondii via PTEN-mediated deactivation of Akt

doi: 10.1371/journal.ppat.1012907

Figure Lengend Snippet: ( A ) CHO cells were challenged with RH- T . gondii . Cell lysates collected at 2 h were probed for Akt and phospho-S473 Akt. Relative density of phospho-Akt was normalized to total Akt and compared to the uninfected sample. Densitometry graph shown as mean + SEM from 3 independent experiments. ( B-C ) CHO cells or primary brain endothelial cells were transfected with plasmids encoding HA-tagged CA- or HA-tagged WT-Akt. Lysates were probed for actin, HA, Akt and phospho-S473 Akt. Cells were challenged with T . gondii and treated with Saracatinib (1 μM). Cell lysates collected at 2 h were probed for Akt and phospho-S473 Akt. Relative density of phospho-Akt and densitometries were examined as above. Percentages of infected cells and tachyzoites per 100 cells were assessed at 24 h. Data are shown as mean ± SEM. The graphs show data from 6 independent monolayers pooled from 3 different experiments. Data points are shown as circles, triangles, or squares corresponding to individual monolayers within each experiment. Significance was determined using two-way unpaired Student’s t test comparing 6 individual monolayers per group (**p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Cells were stained with antibodies against PTEN (Santa Cruz Biotechnology, Dallas, TX) or anti-phospho S473 Akt (Novus, Centennial, CO).

Techniques: Transfection, Infection

Journal: PLOS Pathogens

Article Title: Inhibition of Src signaling induces autophagic killing of Toxoplasma gondii via PTEN-mediated deactivation of Akt

doi: 10.1371/journal.ppat.1012907

Figure Lengend Snippet: B6 mice were infected with 10 ME49 T . gondii tissue cysts for four weeks before treatment with Saracatinib (10 or 15 mg kg -1 oral gavage twice per day; 5 days per week), TMP-SMX (120:600 mg kg -1 oral gavage once a day; 5 days per week) or vehicle. Mice were euthanized after 4 weeks of treatment (8 weeks total infection). ( A-B ) Representative images of retinas and brains of chronically infected mice after four weeks of infection prior to Saracatinib, TMP-SMX, or vehicle administration. Retina: arrowhead = vitreal inflammation; arrow = perivascular inflammation; asterix = disruption of retinal architecture. Brain: arrowhead = perivascular inflammation; arrow, tissue cyst; asterix; microglial nodule. Scale bar, 50 μm. Data represented as mean ± SEM from 8 mice per group pooled from 2–3 independent experiments. (C-D) Representative images of retinas and brains as well as histopathological scoring at 8 weeks post-infection (4 weeks of infection followed by 4 weeks of treatment). Scale bar, 50 μm. Data are shown as mean ± SEM from mice pooled from 2–3 independent experiments: Retina- n = 17 (Ctr), 5 (TMP-SMX), 15 (Saracatinib 10 mg/kg), and 8 (Saracatinib 15 mg/kg). Brain- n = 15 (Ctr), 5 (TMP-SMX), 17 (Saracatinib 10 mg/kg), and 8 (Saracatinib 15 mg/kg). Statistical significance was determined using two-way ANOVA with Tukey multiple comparison test. ( E ) T . gondii B1 gene was examined in the eye qPCR. Levels were compared to those of one control mouse that was given an arbitrary value of 1. Bars represent mean ± SEM pooled from mice pooled from 2–3 independent experiments: n = 11 (Ctr), 4 (TMP-SMX), 10 (Saracatinib 10 mg/kg), and 10 (Saracatinib 15 mg/kg). Statistical significance was determined using one-way ANOVA with Tukey multiple comparison test. ( F ) Number of T . gondii tissue cysts were counted per brain homogenates. Data are shown as mean ± SEM from mice pooled from 2–3 independent experiments: n = 11 (Ctr), 5 (TMP-SMX), 9 (Saracatinib 10 mg/kg), and 8 (Saracatinib 15 mg/kg). Statistical significance was determined using one-way ANOVA with Tukey multiple comparison test. ( G ). Levels of IL-12 p40, IFN-γ, TNF-α and/or NOS2 mRNA in the eye and brain were measured using RT-qPCR. Data shown as mean ± SEM from mice pooled from 2 independent experiments: Retina- n = 6 Ctr, 7 Saracatinib (IFN-γ), 6 per group (TNF-α), 7 Ctr, 6 Saracatinib (IL-12 p40), and 6 per group (NOS2). Brain- n = 5 per group (IFN-γ, TNF-α, IL-12 p40, NOS2). Statistical significance was determined using two-way unpaired t test. (H) Serum levels of IFN-γ, TNF-α and IL-12 p40 were measured by ELISA. Data shown as mean ± SEM from mice pooled from 2 independent experiments: n = 7 per group (IFN-γ), 5 Ctr, 7 Saracatinib (TNF-α), and 6 Ctr, 7 Saracatinib (IL-12 p40). Statistical significance was determined using two-way unpaired t test. ( I ) Serum titers of anti- T . gondii IgG were measured by ELISA and are represented as mean ± SEM from 5 mice per group. Results are representative of 2 independent experiments. ( J , K ) B6 mice infected with ME49 T . gondii tissue cysts for 6 weeks were treated with Saracatinib or vehicle for four days. Brain sections were stained with antibodies against T . gondii , phospho-S473 Akt or PTEN. Scale bar, 10 μm. Data is shown as mean ± SEM from 4 mice. Statistical significance determined using two-way unpaired Student’s t Test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Cells were stained with antibodies against PTEN (Santa Cruz Biotechnology, Dallas, TX) or anti-phospho S473 Akt (Novus, Centennial, CO).

Techniques: Infection, Disruption, Comparison, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining

Journal: BMC Gastroenterology

Article Title: Hepatitis C virus E2 protein involve in insulin resistance through an impairment of Akt/PKB and GSK3β signaling in hepatocytes

doi: 10.1186/1471-230x-12-74

Figure Lengend Snippet: Figure 5 Insulin-induced phosphorylation of Akt and GSK3β was inhibited by E2 protein. After being transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and a serum-free incubation 16 hours, cells were subjected to a insulin stimulation for an indicated time period (0, 15, 30 and 60 minutes) followed by western blotting with indicated antibodies to reveal a time-dependent increase of phosphorylation of Akt (A) and GSK3β (C). Furthermore, transfected or untransfected cells were treated insulin for 30 minutes and then subjected to western blotting to detect Ser473 and Thr308 phosphorylation of Akt (B) serine phosphorylation of GSK3β (D). Results suggested that an expression of E2 inhibited insulin-induced Thr308 phosphorylation of Akt and GSK3β phosphorylation.

Article Snippet: IRS-1, insulin receptor-beta (IRβ), phosphor-Akt (Ser473), ubiquitin, GSK3β and phosphor-GSK3β (Ser9) antibodies were from Cell Signaling Technology. phosphor-Akt (Thr308) antibodies was from Novus Biological.

Techniques: Phospho-proteomics, Transfection, Incubation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

doi: 10.1371/journal.pone.0026581

Figure Lengend Snippet: Adult male C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. (A) Left ventricular tissue lysates were IP with an anti-pY antibody and subjected to in vitro lipid kinase assay. PIP, the phosphorylated end-product. The bar graph shows the densitometric scanning results of the measurement of PI3K activities in the control (n = 6) and ISO-treated (n = 6) mice. (B, C) Representative Western blot analyses were performed on LV tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. The bar graphs show the densitometric scanning results from two individual experiments (n = 6). In another series of experiments, mice were pretreated with vehicle (5% DMSO) or LY294002 (LY, 1.4 mg/kg, i.p.) for 30 min before the ISO treatment. In vitro lipid kinase assay (D) and Western blot analyses (E) were performed as described above. The bar graph shows the densitometric scanning results in the control (n = 6), ISO (n = 6), and LY/ISO (n = 6) groups. In all Western blotting experiments, data were normalized with individual total protein levels. *, p<0.05 versus vehicle control.

Article Snippet: Protein samples were loaded and run in either 4–12% Bis-Tris precast gels or 3–8% Tris-Acetate precast gels (Invitrogen, Carlsbad, CA), blotted onto a PVDF membrane (Bio-Rad, Hercules, CA), and detected by the following antibodies: phospho-Akt (Ser473) antibody (R&D systems, Minneapolis, MN), phospho-Akt (Thr308), total Akt, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-mTOR (Ser2448), total mTOR, phospho-P70S6K (Thr389, Thr421/Ser424), total P70S6K, phospho-S6 (Ser235/236, Ser240/244), total S6, phospho-GSK-3α (Ser21), total GSK-3α, phospho-GSK-3β (Ser9), total GSK-3β, phospho-FOXO1(Thr24)/FOXO3a(Thr32), phospho-FOXO3a (Ser318/321, Ser253), total FOXO1 antibodies (Cell Signaling, Danvers, MA), total FOXO3a and Actin antibodies (Millipore, Billerica, MA).

Techniques: Control, In Vitro, Kinase Assay, Western Blot

Journal: PLoS ONE

Article Title: β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

doi: 10.1371/journal.pone.0026581

Figure Lengend Snippet: Adult male C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for the indicated time. Shown are representative Western blots performed on LV tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-ERK1/2 (Thr202/Tyr204), phospho-P70S6K (Thr389), phospho-P70S6K (Thr421/Ser424), phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), phospho-GSK-3α (Ser21), phospho-GSK-3β (Ser9), and phospho-FOXO3a (Ser318/321). Blots of individual total protein and actin were also included.

Article Snippet: Protein samples were loaded and run in either 4–12% Bis-Tris precast gels or 3–8% Tris-Acetate precast gels (Invitrogen, Carlsbad, CA), blotted onto a PVDF membrane (Bio-Rad, Hercules, CA), and detected by the following antibodies: phospho-Akt (Ser473) antibody (R&D systems, Minneapolis, MN), phospho-Akt (Thr308), total Akt, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-mTOR (Ser2448), total mTOR, phospho-P70S6K (Thr389, Thr421/Ser424), total P70S6K, phospho-S6 (Ser235/236, Ser240/244), total S6, phospho-GSK-3α (Ser21), total GSK-3α, phospho-GSK-3β (Ser9), total GSK-3β, phospho-FOXO1(Thr24)/FOXO3a(Thr32), phospho-FOXO3a (Ser318/321, Ser253), total FOXO1 antibodies (Cell Signaling, Danvers, MA), total FOXO3a and Actin antibodies (Millipore, Billerica, MA).

Techniques: Control, Western Blot

Journal: PLoS ONE

Article Title: β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

doi: 10.1371/journal.pone.0026581

Figure Lengend Snippet: Adult male C57BL/6 mice were treated with vehicle control (saline) or isoproterenol (ISO, 1.25 mg/kg, i.N) for 30 min. (A) Representative Western blot analyses were performed on lung or kidney tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. (B, C) The bar graphs show the densitometric scanning results from two seperate experiments (n = 6). Data are normalized with individual total protein levels and represent means ± S.E. of percent change in protein phosphorylation relative to that of vehicle control. *, p<0.05 versus vehicle control.

Article Snippet: Protein samples were loaded and run in either 4–12% Bis-Tris precast gels or 3–8% Tris-Acetate precast gels (Invitrogen, Carlsbad, CA), blotted onto a PVDF membrane (Bio-Rad, Hercules, CA), and detected by the following antibodies: phospho-Akt (Ser473) antibody (R&D systems, Minneapolis, MN), phospho-Akt (Thr308), total Akt, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-mTOR (Ser2448), total mTOR, phospho-P70S6K (Thr389, Thr421/Ser424), total P70S6K, phospho-S6 (Ser235/236, Ser240/244), total S6, phospho-GSK-3α (Ser21), total GSK-3α, phospho-GSK-3β (Ser9), total GSK-3β, phospho-FOXO1(Thr24)/FOXO3a(Thr32), phospho-FOXO3a (Ser318/321, Ser253), total FOXO1 antibodies (Cell Signaling, Danvers, MA), total FOXO3a and Actin antibodies (Millipore, Billerica, MA).

Techniques: Control, Saline, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

doi: 10.1371/journal.pone.0026581

Figure Lengend Snippet: Adult male C57BL/6 mice were pretreated with vehicle (5% DMSO) or H-89 (20 mg/kg, i.p.) for 30 min before treatment with control (saline) or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. (A) In vitro lipid kinase assay was performed as described. PIP, the phosphorylated end-product. The bar graph shows the densitometric scanning results of the measurement of PI3K activities in the control, ISO-treated and H-89 + ISO-treated mice (n = 4). (B, C) Representative Western blot analyses were performed on LV tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-FOXO1 (Thr24), total Akt and total FOXO. The bar graphs show the densitometric scanning results. Data are normalized with individual total protein levels and represent means ± S.E. of percent change in protein phosphorylation relative to that of the control. *, p<0.05 versus the control.

Article Snippet: Protein samples were loaded and run in either 4–12% Bis-Tris precast gels or 3–8% Tris-Acetate precast gels (Invitrogen, Carlsbad, CA), blotted onto a PVDF membrane (Bio-Rad, Hercules, CA), and detected by the following antibodies: phospho-Akt (Ser473) antibody (R&D systems, Minneapolis, MN), phospho-Akt (Thr308), total Akt, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-mTOR (Ser2448), total mTOR, phospho-P70S6K (Thr389, Thr421/Ser424), total P70S6K, phospho-S6 (Ser235/236, Ser240/244), total S6, phospho-GSK-3α (Ser21), total GSK-3α, phospho-GSK-3β (Ser9), total GSK-3β, phospho-FOXO1(Thr24)/FOXO3a(Thr32), phospho-FOXO3a (Ser318/321, Ser253), total FOXO1 antibodies (Cell Signaling, Danvers, MA), total FOXO3a and Actin antibodies (Millipore, Billerica, MA).

Techniques: Control, Saline, In Vitro, Kinase Assay, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

doi: 10.1371/journal.pone.0026581

Figure Lengend Snippet: C57BL/6 mice were treated with vehicle control, dobutamine (Dob., 1.7 mg/kg, i.p.), or formoterol (For., 2.1 mg/kg, i.p.), for 30 min. (A) In vitro lipid kinase assay was performed as described except with higher amount of LV lysates (1 mg). PIP, the phosphorylated end-product. The bar graph shows the densitometric scanning results of the measurement of PI3K activities in the control, Dob.-treated and For.-treated mice (n = 4). (B) Representative Western blot analyses were performed on left ventricular lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. The bar graphs show the densitometric scanning results from two seperate experiments (n = 5). Data are normalized with individual total protein levels and represent means ± S.E. of percent change in protein phosphorylation relative to that of vehicle control. *, p<0.05 versus vehicle control.

Article Snippet: Protein samples were loaded and run in either 4–12% Bis-Tris precast gels or 3–8% Tris-Acetate precast gels (Invitrogen, Carlsbad, CA), blotted onto a PVDF membrane (Bio-Rad, Hercules, CA), and detected by the following antibodies: phospho-Akt (Ser473) antibody (R&D systems, Minneapolis, MN), phospho-Akt (Thr308), total Akt, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-mTOR (Ser2448), total mTOR, phospho-P70S6K (Thr389, Thr421/Ser424), total P70S6K, phospho-S6 (Ser235/236, Ser240/244), total S6, phospho-GSK-3α (Ser21), total GSK-3α, phospho-GSK-3β (Ser9), total GSK-3β, phospho-FOXO1(Thr24)/FOXO3a(Thr32), phospho-FOXO3a (Ser318/321, Ser253), total FOXO1 antibodies (Cell Signaling, Danvers, MA), total FOXO3a and Actin antibodies (Millipore, Billerica, MA).

Techniques: Control, In Vitro, Kinase Assay, Western Blot, Phospho-proteomics