Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: Insulin stimulates GLUT4 translocation toward the plasma membrane in an Akt1/2-dependent manner. (A) The Akt1 or Akt2 concentration/intensity standard curves were made using a human recombinant Akt1 or Akt2 respectively. 3T3-L1-GLUT4myc fibroblasts on day 14 after differentiation induction were lysed followed by western blotting using antibodies against Akt1 and Akt2, and the amount of Akt1 and Akt2 was calculated from each standard curve ( n =4 independent experiments). (B) 3T3-L1-GLUT4myc adipocytes were treated with insulin (100 nM) in the presence and absence of MK2206 (+MK) (5 μM). Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant. (C) In the left panel, 3T3-L1-GLUT4myc adipocytes were transfected with the NC siRNA or the Akt1/2 siRNA, and 48 h after transfection western blotting was carried out using antibodies against Akt1/2 or β-actin. Signal intensities for Akt1/2 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of Akt1/2 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the Akt1/2 siRNA (Akt1/2 KD) were left untreated or treated with insulin (100 nM) for 20 min. Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Concentration Assay, Recombinant, Western Blot, Transfection, Expressing

Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: Insulin stimulates phosphorylation of Akt1/2 in a PI3K-dependent manner in 3T3-L1-GLUT4myc adipocytes. Cells were left untreated or treated with insulin (Ins) (100 nM) for 10 min in the presence and absence of wortmannin (+WM) (20 nM) (A) or BX912 (+BX) (100 nM) (B). Western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graphs, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Phospho-proteomics, Western Blot

Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PI3K ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PDK1 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

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

Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: PI3K-dependent and PDK1-independent Akt1 phosphorylation and PI3K-/PDK1-dependent Akt2 phosphorylation under cell-free conditions. Akt1 or Akt2 was reacted with (+) and without (−) PI3K (1 μg/ml) (A and C) or PDK1 (1 μg/ml) (B and D) in the presence and absence of wortmannin (WM) (20 nM) or BX912 (BX) (100 nM), and western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1 (pAkt1) or Akt2 (pAkt2) were normalized to those for Akt1 or Akt2. In the graphs, each value represents the mean (± s.e.m .) intensity for pAkt1 or pAkt2 at each site ( n =4). P values, Dunnett's test. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Phospho-proteomics, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Assessing the anti-inflammatory effects of quercetin using network pharmacology and in vitro experiments

doi: 10.3892/etm.2022.11230

Figure Lengend Snippet: Effects of quercetin on the phosphorylation of Akt in LPS-stimulated RAW264.7 cells. (A and B) The cells were pretreated with different concentrations of quercetin (5,10 and 20 µM) for 1 h and then stimulated with LPS (1 µg/ml) for another 6 h. (A) After different treatments, the protein levels of p-Akt, Akt and GAPDH were determined using western blot analysis and (B) the results were statistically analyzed. (C-G) RAW264.7 cells were pretreated with quercetin (20 µM) and AKT inhibitor (MK-2206; 5 µM) for 1 h in 6-well microplates and stimulated with LPS (1 µg/ml) for 6 or 24 h. The (C) mRNA and (D and E) protein expression levels of TNF-α, IL-6 and IL-1β were analyzed using reverse transcription-quantitative PCR and western blot analysis, respectively. (D) Representative western blots and (E) quantified results. (F and G) The protein levels of p-Akt, Akt were analyzed using western blot analysis. (F) Representative western blots and (G) quantified results. ## P<0.01 vs. non-intervention group; ** P<0.01 vs. LPS alone group; & P<0.05, && P<0.01 vs. quercetin treatment group. LPS, lipopolysaccharide; p, phosphorylated.

Article Snippet: Antibodies against phosphorylated (p-)Akt (cat. no. T55561) and Akt (cat. no. T40067) were obtained from Abmart.

Techniques: Phospho-proteomics, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Chest

Article Title: IL-27 Is Elevated in Patients With COPD and Patients With Pulmonary TB and Induces Human Bronchial Epithelial Cells to Produce CXCL10

doi: 10.1378/chest.10-3297

Figure Lengend Snippet: Effects of IL-27 on the activation of the PI3K-Akt signaling pathway in PBEC grown on air-liquid interface culture. PBEC were incubated with or without IL-27 (100 ng/mL) for 5 min. A, Intracellular expression of phosphorylated PI3K. B, Intracellular expression of phosphorylated Akt. The isotypic control represents the cell populations stained with antimouse IgG1 isotype control. C, Activation of PI3K was determined by enzyme-linked immunosorbent assay 15 min after stimulation with IL-27. *** P < .001 vs medium control. D, Phosphorylated Akt and total Akt levels were determined by Western blot analysis 15 min after stimulation with IL-27. The results are from three independent experiments. PI3K = phosphatidylinositol 3-OH kinase. See , legends for expansion of abbreviations.

Article Snippet: For flow cytometric analysis, permeabilized PBEC were stained with mouse antihuman phosphorylated Akt (BD Pharmingen) and phosphorylated PI3K (Cell Signaling Technology) or mouse IgG1 antibodies (BD Pharmingen), followed by fluorescein isothiocyanate-conjugated goat antimouse secondary antibodies.

Techniques: Activation Assay, Incubation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Chest

Article Title: IL-27 Is Elevated in Patients With COPD and Patients With Pulmonary TB and Induces Human Bronchial Epithelial Cells to Produce CXCL10

doi: 10.1378/chest.10-3297

Figure Lengend Snippet: Effects of siRNA-PI3K p110α, siRNA-PI3K p110β, and siRNA-Akt on IL-27-induced CXCL10 expression. BEAS-2B cells were transfected with or without siRNA-PI3K p110α and siRNA-PI3K p110β, and their blocking effects were validated by Western blot. A, Blocking effects on the expression of PI3K p110α. B, Blocking effects on the expression of PI3K p110β. C, Blocking effects on the phosphorylation of Akt. BEAS-2B cells were transfected with siRNA-PI3K p110α, siRNA-PI3K p110β, and siRNA-Akt. The cells and cell supernatants were then harvested at 2 h and 24 h, respectively, after stimulation with 100 ng/mL of IL-27. D, Level of CXCL10 gene expression measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). E, Level of protein production measured by RT-PCR and ELISA. Results are expressed as the arithmetic mean ± SD from three independent experiments. * P < .05, ** P < .01, and *** P < .001 when compared between groups denoted by horizontal lines. GADPH = glyceraldehyde 3-phosphate dehydrogenase. See , legends for expansion of other abbreviations.

Article Snippet: For flow cytometric analysis, permeabilized PBEC were stained with mouse antihuman phosphorylated Akt (BD Pharmingen) and phosphorylated PI3K (Cell Signaling Technology) or mouse IgG1 antibodies (BD Pharmingen), followed by fluorescein isothiocyanate-conjugated goat antimouse secondary antibodies.

Techniques: Expressing, Transfection, Blocking Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay