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: Journal of Virology

Article Title: Peste des Petits Ruminants Virus-Induced Novel MicroRNA miR-3 Contributes To Inhibit Type I IFN Production by Targeting IRAK1

doi: 10.1128/JVI.02045-20

Figure Lengend Snippet: Novel miR-3 enhances the infection and progeny of PPRV by suppression of IFN-α production. (A) Goat PBMCs were transfected with miR-3 inhibitor or control RNA (IC) at a final concentration of 100 nM. Twenty-four hours after transfection, the cells were infected with PPRV (MOI = 1) for 2 h and then washed. Cells were harvested at the indicated hour postinfection (hpi), and the indicated protein levels in the IC and miR-3 inhibitor groups were determined by Western blotting. (B to D) Goat PBMCs were transfected with IC or miR-3 inhibitor at a final concentration of 100 nM and 48 h later the cells were infected with PPRV at an MOI of 1. The virus titers in the supernatants and the indicated ISG expression at the indicated time points postinfection were measured by TCID50 assay (B) and qRT-PCR (C and D), respectively. (E and F) Goat PBMCs were cotransfected with miR-3 inhibitor or control RNA and siRNA against IRAK1. After 24 h, the cells were infected with PPRV for 24 h, and protein levels as indicated and virus titers in the supernatants were determined by Western blotting (E) and TCID50 (F), respectively. (G to I) Goat PBMCs were transfected with miR-3 mimic or mimic control (MC) and further treated with or without IFN-α (100 U/ml). After 24 h, the cells were infected with PPRV (MOI = 1) for 24 h, and PPRV V and N protein levels (G) and virus titers (H) in the supernatants were determined by Western blotting and TCID50 assay, respectively. The ISGs indicated were also examined by qRT-PCR (I). (J and K) Goat PBMCs were transfected with novel miR-3 mimic or mimic control. After 24 h, the cells were infected with PPRV (MOI = 1) for 24 h, and IRF3 mRNA (J) and phosphorylation levels (K) were determined by qRT-PCR and Western blot assay, respectively. (L) Goat PBMCs were transfected with nonspecific control siRNA and siRNA against IRF3. After 24 h, IRF3 protein levels were determined by Western blotting and normalized to GAPDH. (M and N) Goat PBMCs were transfected with miR-3 mimic or mimic control and further transfected with siRNA against IRF3 or control siRNA. After 24 h, the cells were infected with PPRV (MOI = 1) for 24 h, and IRF3 phosphorylation (M) and indicated ISG mRNA expression (N) were determined by Western blotting and qRT-PCR assay, respectively. GAPDH was used as a loading control in qRT-PCR and Western blot analysis. Results are expressed as means ± standard error of mean (SEM). P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control; *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: Membranes were probed overnight at 4°C with an anti-PPRV-N or anti-PPRV-V monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), a rabbit polyclonal antibody against mouse IL-10 (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFN-α (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFNG (1:1,500; Abcam), or a rabbit polyclonal antibody against mouse TNF-α (1:1,500; Abcam).

Techniques: Infection, Transfection, Concentration Assay, Western Blot, Expressing, TCID50 Assay, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Peste des Petits Ruminants Virus-Induced Novel MicroRNA miR-3 Contributes To Inhibit Type I IFN Production by Targeting IRAK1

doi: 10.1128/JVI.02045-20

Figure Lengend Snippet: Novel miR-3 inhibits IFN-α production during PPRV infection. (A to D) Goat PBMCs were transfected with control mimic (MC) and different concentrations of miR-3 mimic (A and B), or else control inhibitor (IC) and miR-3 inhibitor (C and D), and 48 h later the cells were infected with PPRV at an MOI of 1. Twenty-four hours later, the mRNA (A and C) and protein levels (B and D) of cytokines indicated were measured by qRT-PCR and Western blotting, respectively. (E and F) Goat PBMCs were transfected with control mimic (MC), miR-3 mimic, control inhibitor (IC), or miR-3 inhibitor for 48 h, and then the cells were stimulated with poly(I·C). Twenty-four hours later, IFN-α mRNA (E) and protein expression (F) were measured by qRT-PCR and Western blot assay, respectively. GAPDH was used as a loading control in qRT-PCR and Western blot analysis. Results are expressed as means ± standard error of mean (SEM). P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control; *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: Membranes were probed overnight at 4°C with an anti-PPRV-N or anti-PPRV-V monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), a rabbit polyclonal antibody against mouse IL-10 (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFN-α (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFNG (1:1,500; Abcam), or a rabbit polyclonal antibody against mouse TNF-α (1:1,500; Abcam).

Techniques: Infection, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Peste des Petits Ruminants Virus-Induced Novel MicroRNA miR-3 Contributes To Inhibit Type I IFN Production by Targeting IRAK1

doi: 10.1128/JVI.02045-20

Figure Lengend Snippet: Regulation of IFN-α production by novel miR-3 is mediated by IRAK1. (A and B) Goat PBMCs were transfected with nonspecific control siRNA and siRNA against IRAK1. After 24 h, IRAK1 mRNA (A) and protein levels (B) were determined by qRT-PCR and Western blotting, respectively, and normalized to GAPDH. (C to E) Goat PBMCs were cotransfected with novel miR-3 inhibitor or control RNA and siRNA against IRAK1. After 24 h, the cells were stimulated with poly(I·C) for 24 h, and IFN-α mRNA (C), protein expression (D), and secretion (E) were determined by qRT-PCR, Western blotting, and ELISA, respectively. (F) Goat PBMCs were cotransfected with novel miR-3 inhibitor or control RNA and siRNA against IRAK1. After 24 h, the cells were stimulated with poly(I·C) for 24 h, and interferon-stimulated genes (ISGs) as indicated were determined by qRT-PCR. GAPDH was used as a loading control in qRT-PCR and Western blot analysis. Results are expressed as means ± standard error of mean (SEM). P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control; *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: Membranes were probed overnight at 4°C with an anti-PPRV-N or anti-PPRV-V monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), a rabbit polyclonal antibody against mouse IL-10 (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFN-α (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFNG (1:1,500; Abcam), or a rabbit polyclonal antibody against mouse TNF-α (1:1,500; Abcam).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Peste des Petits Ruminants Virus-Induced Novel MicroRNA miR-3 Contributes To Inhibit Type I IFN Production by Targeting IRAK1

doi: 10.1128/JVI.02045-20

Figure Lengend Snippet: Novel miR-3 correlates with PPRV infection in goat and sheep. (A) The kinetic levels of PPRV V and N protein in PPRV-infected PBMCs from goat and sheep were determined by Western blotting assay. (B and C) The kinetic levels of miR-3 expression (B) and IFN-α production (C) in PPRV-infected PBMCs from goat and sheep were evaluated by qRT-PCR and ELISA, respectively (n = 25). GAPDH was used as a loading control in Western blotting for PPRV V and N protein analysis. P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control; *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: Membranes were probed overnight at 4°C with an anti-PPRV-N or anti-PPRV-V monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), a rabbit polyclonal antibody against mouse IL-10 (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFN-α (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFNG (1:1,500; Abcam), or a rabbit polyclonal antibody against mouse TNF-α (1:1,500; Abcam).

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Peste des Petits Ruminants Virus-Induced Novel MicroRNA miR-3 Contributes To Inhibit Type I IFN Production by Targeting IRAK1

doi: 10.1128/JVI.02045-20

Figure Lengend Snippet: Proposed model for the activation of novel miR-3 expression upon PPRV infection and the role of novel miR-3 in the regulation of the antiviral activity of IFN-α. PPRV V protein alone can activate novel miR-3 expression during PPRV infection, and the NF-κB and p38 pathways may be involved in the upregulation of miR-3. Novel miR-3 interferes with IFN-α production by repressing IRAK1 expression, leading to viral escape from the host innate-immunity response. ISGs can be stimulated in an IRF3-dependent manner during PPRV infection.

Article Snippet: Membranes were probed overnight at 4°C with an anti-PPRV-N or anti-PPRV-V monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), a rabbit polyclonal antibody against mouse IL-10 (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFN-α (1:1,500; ABclonal), a rabbit polyclonal antibody against mouse IFNG (1:1,500; Abcam), or a rabbit polyclonal antibody against mouse TNF-α (1:1,500; Abcam).

Techniques: Activation Assay, Expressing, Infection, Activity Assay

Journal: Cell Death & Disease

Article Title: Role of the androgen receptor in melanoma aggressiveness

doi: 10.1038/s41419-025-07350-4

Figure Lengend Snippet: A , B Frequency of MICA/B in untreated (white bars) or R1881-treated (black bars) AMM16 cells. B Data from three independent experiments are represented as mean ± SD. n = 3; ** p -value < 0.01. C AMM16 were untreated or treated for 6 h with R1881, in absence or presence of bicalutamide (Bic). When indicated, cells were pretreated with MG132. WB using the indicated antibodies was done. D Quiescent AMM16 cells on coverslips were left untreated or treated for 6 h with R1881. They were stained for MICA/B. Images captured by confocal microscope show the staining of MICA/B (green) and nuclei (blu). Merged images are presented in the right panels. They are representative of three independent experiments. Bar, 10 μm. E Cells were unstimulated or stimulated with R1881, in the absence or presence of Bic for 6 h. sMICA was assayed and quantified by ELISA. Graph is representative of three independent experiments; * p -value < 0.05. F , G NK cells were untreated or treated with AMM16 cells derived CM, as indicated. F WB with the antibodies against the indicated proteins was done. G NK cells were stained as reported in Methods section. Images captured by confocal microscopy show the staining of NKG2D (red) and nuclei (blu). Merged images are presented in the right panels. They are representative of three independent experiments. Bar, 2.5 μm.

Article Snippet: NK cells fixed with paraformaldehyde (4%, w/v, in PBS), were permeabilized using Triton-X100 (0,1%, w/v Biorad in H 2 O) and incubated overnight with diluted (1:30 in PBS) rabbit polyclonal anti-NKG2D antibody (KLRK1-A11964, ABclonal, Düsseldorf, Germany).

Techniques: Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Derivative Assay, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Role of the androgen receptor in melanoma aggressiveness

doi: 10.1038/s41419-025-07350-4

Figure Lengend Snippet: Androgen stimulation of melanoma cells triggers the assembly of the AR/ADAM10/β1 integrin complex. Such complex enables ADAM10 to act as MICA sheddase, on one hand. The MICA shedding, hence, causes the effective masking from NKG2D recognition allowing the immune-escape. On the other, the complex mediates the androgen-induced invasiveness of melanoma cells through the activation of FAK and ERK.

Article Snippet: NK cells fixed with paraformaldehyde (4%, w/v, in PBS), were permeabilized using Triton-X100 (0,1%, w/v Biorad in H 2 O) and incubated overnight with diluted (1:30 in PBS) rabbit polyclonal anti-NKG2D antibody (KLRK1-A11964, ABclonal, Düsseldorf, Germany).

Techniques: Activation Assay