Journal: RSC Advances

Article Title: miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1

doi: 10.1039/c8ra06866f

Figure Lengend Snippet: Downregulation in the FOXO1 pSer256/total FOXO1 ratio and AKT1 pSer473 level caused by ox-LDL treatment in HUVECs or HAECs could be partially rescued by miR-183-5p, miR-96-5p or miR-182-5p agomir treatment, which doesn’t affect the AKT1 total protein expression level (A–E). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: For western blot analysis, primary antibodies against FOXO1 (NB100-2312), FOXO1-pSer256 (NB100-81927), AKT1 (NBP1-51602), AKT1-pSer473 (NB100-56749), and GAPDH (NB100-56875) were purchased from Bio-techne (Shanghai, China).

Techniques: Expressing

Journal: RSC Advances

Article Title: miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1

doi: 10.1039/c8ra06866f

Figure Lengend Snippet: AKT1 overexpression significantly increased the AKT1 protein expression level and Ser473 phosphorylation level while reducing FOXO1 protein expression in HUVECs or HAECs with or without ox-LDL treatment (A–G). *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: For western blot analysis, primary antibodies against FOXO1 (NB100-2312), FOXO1-pSer256 (NB100-81927), AKT1 (NBP1-51602), AKT1-pSer473 (NB100-56749), and GAPDH (NB100-56875) were purchased from Bio-techne (Shanghai, China).

Techniques: Over Expression, Expressing

Journal: RSC Advances

Article Title: miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1

doi: 10.1039/c8ra06866f

Figure Lengend Snippet: AKT1 overexpression partially reduced ox-LDL-induced apoptosis in HUVECs or HAECs by upregulating miR-183-5p and miR-182-5p. (A–F) miR-183-5p and miR-182-5p were upregulated by AKT1 overexpression in HUVECs or HAECs with or without ox-LDL treatment, while miR-96-5p was not significantly influenced. (G and H) Antagomir treatment reducing the endogenous miR-183-5p or miR-182-5p expression levels partially reduced the anti-apoptotic effect of AKT1 overexpression in the HUVECs or HAECs treated by ox-LDL. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: For western blot analysis, primary antibodies against FOXO1 (NB100-2312), FOXO1-pSer256 (NB100-81927), AKT1 (NBP1-51602), AKT1-pSer473 (NB100-56749), and GAPDH (NB100-56875) were purchased from Bio-techne (Shanghai, China).

Techniques: Over Expression, 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