pgsk3β Search Results


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  • 99
    Millipore pgsk3β s389
    Pgsk3β S389, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc pgsk3β
    Phosphorylation of Akt, GSK3β, mTOR and Foxo3a in myocardium from FVB and ALDH2 mice treated with or without streptozotocin . (A) pAkt-to-Akt ratio; (B) <t>pGSK3β-to-GSK3β</t> ratio; (C) pmTOR-to-mTOR ratio; (D) pFoxo3a-to-Foxo3a ratio. Insets: representative gel blots of pan and phosphorylated Akt, GSK3β, mTOR and Foxo3a (GAPDH as loading control) using specific antibodies. Mean ± SEM, n = 6 to 7 mice per group. * P
    Pgsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson pgsk3β
    Colocalization of tau phosphorylated at threonine 175 (pThr175 tau) and tau phosphorylated at threonine 231 (pThr 231 tau) Hippocampal neurons in chronic traumatic encephalopathy (CTE) (upper panels) demonstrate colocalization of pThr 175 tau and pThr 231 tau. The presence of pathologic tau oligomers (T22 immunoreactivity) was colocalized to pThr 231 tau-immunoreactive neurons in CTE (middle panels). Consistent with a role in activation of glycogen synthase kinase–3β (GSK3β) in inducing pathologic tau deposition, we observed the colocalization of pThr 175 tau with the active <t>pGSK3β</t> immunoreactivity (lower panel). Tissues were immunolabeled with rabbit anti-pThr 175 tau, rabbit anti-pThr 231 tau, rabbit anti-T22 antibody, and mouse anti-GSK3β pTyr 216 antibody. For double-labeled tissue, red channel antibodies were labeled directly with Alexa Fluor 555. Scale bar = 5 μm.
    Pgsk3β, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc pgsk3β y216
    Colocalization of tau phosphorylated at threonine 175 (pThr175 tau) and tau phosphorylated at threonine 231 (pThr 231 tau) Hippocampal neurons in chronic traumatic encephalopathy (CTE) (upper panels) demonstrate colocalization of pThr 175 tau and pThr 231 tau. The presence of pathologic tau oligomers (T22 immunoreactivity) was colocalized to pThr 231 tau-immunoreactive neurons in CTE (middle panels). Consistent with a role in activation of glycogen synthase kinase–3β (GSK3β) in inducing pathologic tau deposition, we observed the colocalization of pThr 175 tau with the active <t>pGSK3β</t> immunoreactivity (lower panel). Tissues were immunolabeled with rabbit anti-pThr 175 tau, rabbit anti-pThr 231 tau, rabbit anti-T22 antibody, and mouse anti-GSK3β pTyr 216 antibody. For double-labeled tissue, red channel antibodies were labeled directly with Alexa Fluor 555. Scale bar = 5 μm.
    Pgsk3β Y216, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc α pgsk3β
    The effects of neonatal LPS on Nrf2 regulating kinases AKT and GSK-3β and tyrosine hydroxylase (TH) and in substantia nigra of PND70 rats and the effects of a Spirulina enriched diet. Densitometric analyses of phosphoAKT (pAKT), phospho-GSK3β <t>(pGSK3β)</t> and TH protein expression are shown in Fig.4 A, C, E. Data are plotted as ratio of the pAKT/total AKT (Fig. 4A), pGSK3β/total GSK3β (Fig. 4C) and TH/tubulin (Fig. 4E). Representative blots are shown in Fig. 4 B, D, F; respectively. Two-way ANOVA showed a statistically significant interaction between diet and LPS treatment for: phospho-Akt in Fig. 4A, p= 0.004 and for phospho-GSK3β in Fig. 4C, p=0.0320; however the two-way ANOVA did not show statistically significant interaction between diet and treatment for TH. Bonferroni post-hoc analysis comparing Saline (n = 10) vs LPS (n = 9) and LPS vs LPS+SP0.1% (n = 12) groups are shown, *denotes statistical significance (*p
    α Pgsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc pgsk3β ser9
    The effects of neonatal LPS on Nrf2 regulating kinases AKT and GSK-3β and tyrosine hydroxylase (TH) and in substantia nigra of PND70 rats and the effects of a Spirulina enriched diet. Densitometric analyses of phosphoAKT (pAKT), phospho-GSK3β <t>(pGSK3β)</t> and TH protein expression are shown in Fig.4 A, C, E. Data are plotted as ratio of the pAKT/total AKT (Fig. 4A), pGSK3β/total GSK3β (Fig. 4C) and TH/tubulin (Fig. 4E). Representative blots are shown in Fig. 4 B, D, F; respectively. Two-way ANOVA showed a statistically significant interaction between diet and LPS treatment for: phospho-Akt in Fig. 4A, p= 0.004 and for phospho-GSK3β in Fig. 4C, p=0.0320; however the two-way ANOVA did not show statistically significant interaction between diet and treatment for TH. Bonferroni post-hoc analysis comparing Saline (n = 10) vs LPS (n = 9) and LPS vs LPS+SP0.1% (n = 12) groups are shown, *denotes statistical significance (*p
    Pgsk3β Ser9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam pgsk3β
    The effects of neonatal LPS on Nrf2 regulating kinases AKT and GSK-3β and tyrosine hydroxylase (TH) and in substantia nigra of PND70 rats and the effects of a Spirulina enriched diet. Densitometric analyses of phosphoAKT (pAKT), phospho-GSK3β <t>(pGSK3β)</t> and TH protein expression are shown in Fig.4 A, C, E. Data are plotted as ratio of the pAKT/total AKT (Fig. 4A), pGSK3β/total GSK3β (Fig. 4C) and TH/tubulin (Fig. 4E). Representative blots are shown in Fig. 4 B, D, F; respectively. Two-way ANOVA showed a statistically significant interaction between diet and LPS treatment for: phospho-Akt in Fig. 4A, p= 0.004 and for phospho-GSK3β in Fig. 4C, p=0.0320; however the two-way ANOVA did not show statistically significant interaction between diet and treatment for TH. Bonferroni post-hoc analysis comparing Saline (n = 10) vs LPS (n = 9) and LPS vs LPS+SP0.1% (n = 12) groups are shown, *denotes statistical significance (*p
    Pgsk3β, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Upstate Biotechnology Inc pgsk3β
    The effects of neonatal LPS on Nrf2 regulating kinases AKT and GSK-3β and tyrosine hydroxylase (TH) and in substantia nigra of PND70 rats and the effects of a Spirulina enriched diet. Densitometric analyses of phosphoAKT (pAKT), phospho-GSK3β <t>(pGSK3β)</t> and TH protein expression are shown in Fig.4 A, C, E. Data are plotted as ratio of the pAKT/total AKT (Fig. 4A), pGSK3β/total GSK3β (Fig. 4C) and TH/tubulin (Fig. 4E). Representative blots are shown in Fig. 4 B, D, F; respectively. Two-way ANOVA showed a statistically significant interaction between diet and LPS treatment for: phospho-Akt in Fig. 4A, p= 0.004 and for phospho-GSK3β in Fig. 4C, p=0.0320; however the two-way ANOVA did not show statistically significant interaction between diet and treatment for TH. Bonferroni post-hoc analysis comparing Saline (n = 10) vs LPS (n = 9) and LPS vs LPS+SP0.1% (n = 12) groups are shown, *denotes statistical significance (*p
    Pgsk3β, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgsk3β/product/Upstate Biotechnology Inc
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    91
    Santa Cruz Biotechnology pgsk3β
    BCR affinity promotes BAFF induced PI3K signaling. ( a,b ) HEL TG B cells were left unactivated (Medium), or stimulated with 100 ng/ml BAFF alone (BAFF only) or in combination with 100 ng/ml HEL, HEL2x or HEL3x in the presence or absence of ( a ) the Pan-PI3K inhibitor Ly249002 or ( b ) the PI3Kδ inhibitor CAL-101. After 48 h viability and Mcl-1 protein levels were assessed by flow cytometry (n = 3). ( c ) HEL TG B cells were stimulated with 100 ng/ml BAFF in combination with 100 ng/ml HEL, HEL2x or HEL3x. After 48 h, cells were deprived from stimuli for 3 h, followed by stimulation with 500 ng/ml BAFF. pAkt T308 levels were measured by intracellular flow cytometry at the indicated time points (n = 3). ( d ) HEL TG B cells were activated with the indicated stimuli. Mcl-1 and <t>pGSK3β</t> levels were determined by western blot. β-actin was used as a loading control. Quantification shows fold induction over the signal on day 0, normalized for β-actin. ( e ) HEL TG B cells were left inactivated (Medium), or stimulated with 100 ng/ml BAFF alone (BAFF only) or in combination with 100 ng/ml HEL, HEL2x or HEL3x in the presence of solvent only (DMSO)or the GSK3 inhibitors XXVI or CHIR99021 (CHIR). All cells were cultured in the presence of 1 μM QVD to exclude caspase-mediated Mcl-1 breakdown. After 24 h Mcl-1 expression was assessed by flow cytometry (n = 3). Values show means ± sem. GMI = Geometric Mean Intensity *P
    Pgsk3β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Beyotime pgsk3β
    Effect of HCV infection on insulin-induced phosphorylation of Akt and GSK3β. Huh7.5.1 cells were seeded in a 24-well plate and subjected to serum-free incubation for 20 h, followed by insulin stimulation for the indicated time (0, 15, 30, and 60 min). Western blotting with indicated antibodies revealed a time-dependent change in the phosphorylation of Akt (a) and GSK3β (b) . Furthermore, Huh7.5.1 cells and HCV-infected cells were stimulated with 100 nM insulin for 30 min or left untreated. Extracted proteins were analyzed with antibodies against pAkt, Akt, <t>pGSK3β,</t> and GSK3β (c) .
    Pgsk3β, supplied by Beyotime, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore pgsk3β
    Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. A: Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. HCT116 cells were transfected with dnIκB, dnAKT or dnTCF4 and were treated with IL-1β or TNFα, or were cultured with THP1 macrophages as indicated. The levels of Snail and c-jun were determined by immunoblotting. B: The levels of Snail and <t>pGSK3β</t> were determined by immunoblotting in Hke-3 cells that were treated with IL-1β, TNFα or were co-cultured with macrophages in the absence or the presence of LY 294002 for 24 hours. C : HCT116 and Hke-3 cells were treated with TRAIL in the absence or the presence of IL-1β and LY294002, as indicated. The experiment was performed three times.
    Pgsk3β, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology pgsk3β ser9
    Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. A: Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. HCT116 cells were transfected with dnIκB, dnAKT or dnTCF4 and were treated with IL-1β or TNFα, or were cultured with THP1 macrophages as indicated. The levels of Snail and c-jun were determined by immunoblotting. B: The levels of Snail and <t>pGSK3β</t> were determined by immunoblotting in Hke-3 cells that were treated with IL-1β, TNFα or were co-cultured with macrophages in the absence or the presence of LY 294002 for 24 hours. C : HCT116 and Hke-3 cells were treated with TRAIL in the absence or the presence of IL-1β and LY294002, as indicated. The experiment was performed three times.
    Pgsk3β Ser9, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology pgsk3β y216
    Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. A: Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. HCT116 cells were transfected with dnIκB, dnAKT or dnTCF4 and were treated with IL-1β or TNFα, or were cultured with THP1 macrophages as indicated. The levels of Snail and c-jun were determined by immunoblotting. B: The levels of Snail and <t>pGSK3β</t> were determined by immunoblotting in Hke-3 cells that were treated with IL-1β, TNFα or were co-cultured with macrophages in the absence or the presence of LY 294002 for 24 hours. C : HCT116 and Hke-3 cells were treated with TRAIL in the absence or the presence of IL-1β and LY294002, as indicated. The experiment was performed three times.
    Pgsk3β Y216, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc gsk3β pgsk3β
    MiR-371b-5p utilizes PTEN to orchestrate the PI3K/Akt signaling in hiPSC-ATIICs. a The predicted RNA duplexes of miR-371b-5p target site in the 3′UTR of PTEN ( top panel ). The underlined nucleotides ( UUUGA ) in the target site ( top panel ) were replaced with GGGUC in the mutant 3′UTR construct ( bottom panel ). b Schematic structure of dual-luciferase miRNA target reporter, in which wt or mutant PTEN 3′UTR was inserted into the Pmel and XbaI sites downstream of firefly luciferase (Luc2). c Normalized firefly luciferase activity using the dual-luciferase miRNA target reporter with a wt or mutant PTEN 3′UTR in hiPSC-ATIICs co-transfected with miR-371b-5p mimic. d QRT-PCR analysis of mRNA expression of PTEN in hiPSC-ATIICs transfected with various doses of miR-371b-5p mimic as indicated. The expression levels were normalized to 18 s and presented as percentages of control ( left panel ). PTEN protein expression of each group of miR-371b-5p mimic-transfected hiPSC-ATIICs was analyzed by Western blot using GAPDH as an internal control ( right panel ). e The miR-371b-5p mimic-transfected hiPSC-ATIICs were incubated for 24 hours with and without Rosiglitazone (20 μM). The protein extracts were prepared for Western blot analysis of PTEN, Akt/pAkt, <t>GSK3β/pGSK3β,</t> FOXO3/pFOXO3, and FOXO1/pFOXO1 with GAPDH as control ( left panel ), and the number of viable cells in the cultures was counted 2 days after transfection ( right panel ). ATIICs alveolar epithelial type II cells
    Gsk3β Pgsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk3β pgsk3β/product/Cell Signaling Technology Inc
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    88
    Cell Signaling Technology Inc rabbit anti pgsk3β
    The functional properties of FAK/Wnt signaling pathways in the maintenance of the biological characteristics in the LSC cultured in ESC-CM system. (A) The expression levels of pFAK, FAK, pAkt, Akt, <t>pGSK3β,</t> GSK3β and p21 in the ESC-CM, ESC-CM+FAK inhibitor14 ESC-CM+GSK3β inhibitor or ESC-CM+SiRNA-TERT group. (B) The expression level of β-catenin of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. (C) MTT proliferation assay of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. Data are expressed as the mean±SD (n = 3;*,p
    Rabbit Anti Pgsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc pgsk3β serine 9
    The functional properties of FAK/Wnt signaling pathways in the maintenance of the biological characteristics in the LSC cultured in ESC-CM system. (A) The expression levels of pFAK, FAK, pAkt, Akt, <t>pGSK3β,</t> GSK3β and p21 in the ESC-CM, ESC-CM+FAK inhibitor14 ESC-CM+GSK3β inhibitor or ESC-CM+SiRNA-TERT group. (B) The expression level of β-catenin of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. (C) MTT proliferation assay of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. Data are expressed as the mean±SD (n = 3;*,p
    Pgsk3β Serine 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phosphorylation of Akt, GSK3β, mTOR and Foxo3a in myocardium from FVB and ALDH2 mice treated with or without streptozotocin . (A) pAkt-to-Akt ratio; (B) pGSK3β-to-GSK3β ratio; (C) pmTOR-to-mTOR ratio; (D) pFoxo3a-to-Foxo3a ratio. Insets: representative gel blots of pan and phosphorylated Akt, GSK3β, mTOR and Foxo3a (GAPDH as loading control) using specific antibodies. Mean ± SEM, n = 6 to 7 mice per group. * P

    Journal: BMC Medicine

    Article Title: Mitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3? and mitochondrial function

    doi: 10.1186/1741-7015-10-40

    Figure Lengend Snippet: Phosphorylation of Akt, GSK3β, mTOR and Foxo3a in myocardium from FVB and ALDH2 mice treated with or without streptozotocin . (A) pAkt-to-Akt ratio; (B) pGSK3β-to-GSK3β ratio; (C) pmTOR-to-mTOR ratio; (D) pFoxo3a-to-Foxo3a ratio. Insets: representative gel blots of pan and phosphorylated Akt, GSK3β, mTOR and Foxo3a (GAPDH as loading control) using specific antibodies. Mean ± SEM, n = 6 to 7 mice per group. * P

    Article Snippet: Antibodies for GSK3β, pGSK3β, Akt, pAkt, Foxo3a, pFoxo3a, mTOR, pmTOR, PTEN and pPTEN were purchased from Cell Signaling Technology (Beverly, MA, USA) while antibody for PGC-1α was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless otherwise indicated.

    Techniques: Mouse Assay

    Levels of phosphorylated AKT and GSK3β are significantly decreased in MTLE compared to control. (A) Representative Western blot shows decreased pAKT, pGSK3β-S9 in epilepsy ( n = 7) versus control ( n = 4) tissues. Arrow indicates the case with out sclerosis. Membranes containing 20 μg/lane of total protein were probed with anti-pAKT serine 473 (ser273), total AKT, pGSK3β serine 9 (ser9), pGSK3β tyrosine 216 (tyr216), total GSK3β antibodies and GAPDH antibody as a loading control. (B) Graphic representation of the ratio of pAKT to total AKT (** p = 0.0019), and pGSK3β-S9 to total GSK3β ( p , 0.0001) normalized to GAPDH by unpaired t-test. No significant differences (n.s.) were observed in levels of pGSK3β at tyrosine 216 (Tyr216).

    Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

    Article Title: Dysregulation of PINCH signaling in mesial temporal epilepsy

    doi: 10.1016/j.jocn.2016.10.012

    Figure Lengend Snippet: Levels of phosphorylated AKT and GSK3β are significantly decreased in MTLE compared to control. (A) Representative Western blot shows decreased pAKT, pGSK3β-S9 in epilepsy ( n = 7) versus control ( n = 4) tissues. Arrow indicates the case with out sclerosis. Membranes containing 20 μg/lane of total protein were probed with anti-pAKT serine 473 (ser273), total AKT, pGSK3β serine 9 (ser9), pGSK3β tyrosine 216 (tyr216), total GSK3β antibodies and GAPDH antibody as a loading control. (B) Graphic representation of the ratio of pAKT to total AKT (** p = 0.0019), and pGSK3β-S9 to total GSK3β ( p , 0.0001) normalized to GAPDH by unpaired t-test. No significant differences (n.s.) were observed in levels of pGSK3β at tyrosine 216 (Tyr216).

    Article Snippet: Antibodies included: PINCH1, (BD, Rockville, MD, USA), pAKT serine 473 (Cell Signaling, Danvers, MA, USA), total AKT (Cell Signaling), pGSK3β-S9, pGSK3β-Y216 (Cell Signaling), total GSK3β (SCBT, Santa Cruz, CA, USA), GAPDH (1: 5,000) (SCBT), or Grb-2 (Cell Signaling).

    Techniques: Western Blot

    Scheme summarizing the proposed mechanism by which VPA decreases NFκB/COX2/iNOS-mediated neuroinflammation and pAKT/pGSK-3β-mediated neuroapoptosis in a rat model of CCI.

    Journal: Scientific Reports

    Article Title: Valproate reduces neuroinflammation and neuronal death in a rat chronic constriction injury model

    doi: 10.1038/s41598-018-34915-5

    Figure Lengend Snippet: Scheme summarizing the proposed mechanism by which VPA decreases NFκB/COX2/iNOS-mediated neuroinflammation and pAKT/pGSK-3β-mediated neuroapoptosis in a rat model of CCI.

    Article Snippet: The primary antibodies of GSK-3β, pGSK-3β (1:500 dilution, Cell Signaling Technology, Danvers MA), AKT (1:1000 dilution, Cell Signaling Technology), pAKT (Ser473, 1:500 dilution, Cell Signaling Technology), pNFκB (1:500 dilution, Cell Signaling Technology), COX-2 (1:500 dilution, Abcam London, UK), iNOS (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz CA) and β-actin (1:2000 dilution, Sigma-Aldrich, Saint Louis MO) were used in this experiment.

    Techniques:

    Effects of valproate (VPA, 300 mg/kg/day, i.p.) on the expression of pAKT/AKT and pGSK-3β/GSK-3β proteins in the sciatic nerve (SN), dorsal root ganglia (DRG) and spinal cord (SC) induced by chronic constriction injury (CCI). ( A ) Western blots for pAKT/AKT and pGSK-3β/GSK-3β proteins from the SN, DRG and SC. ( B ) The band intensity was quantified by densitometry and indicated as the relative percentage change compared to the sham group. Data represent the mean ± SEM for 6 rats per group. # p

    Journal: Scientific Reports

    Article Title: Valproate reduces neuroinflammation and neuronal death in a rat chronic constriction injury model

    doi: 10.1038/s41598-018-34915-5

    Figure Lengend Snippet: Effects of valproate (VPA, 300 mg/kg/day, i.p.) on the expression of pAKT/AKT and pGSK-3β/GSK-3β proteins in the sciatic nerve (SN), dorsal root ganglia (DRG) and spinal cord (SC) induced by chronic constriction injury (CCI). ( A ) Western blots for pAKT/AKT and pGSK-3β/GSK-3β proteins from the SN, DRG and SC. ( B ) The band intensity was quantified by densitometry and indicated as the relative percentage change compared to the sham group. Data represent the mean ± SEM for 6 rats per group. # p

    Article Snippet: The primary antibodies of GSK-3β, pGSK-3β (1:500 dilution, Cell Signaling Technology, Danvers MA), AKT (1:1000 dilution, Cell Signaling Technology), pAKT (Ser473, 1:500 dilution, Cell Signaling Technology), pNFκB (1:500 dilution, Cell Signaling Technology), COX-2 (1:500 dilution, Abcam London, UK), iNOS (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz CA) and β-actin (1:2000 dilution, Sigma-Aldrich, Saint Louis MO) were used in this experiment.

    Techniques: Expressing, Western Blot

    Double immunofluorescent staining for S100 (Schwann cell marker), GFAP (DRG satellite cell marker), OX-42 (spinal microglia marker) and pGSK-3β in the sciatic nerve (SN), dorsal root ganglia (DRG) and spinal cord (SC) on day 7 after chronic constriction injury (CCI). pGSK-3β protein expression was significantly increased in the CCI group compared to sham and CCI + VPA groups. Administration of VPA attenuated CCI-enhanced pGSK-3β proteins. Quantitative fluorescence data are depicted. Data represent the mean ± SEM for 6 rats per group. # p

    Journal: Scientific Reports

    Article Title: Valproate reduces neuroinflammation and neuronal death in a rat chronic constriction injury model

    doi: 10.1038/s41598-018-34915-5

    Figure Lengend Snippet: Double immunofluorescent staining for S100 (Schwann cell marker), GFAP (DRG satellite cell marker), OX-42 (spinal microglia marker) and pGSK-3β in the sciatic nerve (SN), dorsal root ganglia (DRG) and spinal cord (SC) on day 7 after chronic constriction injury (CCI). pGSK-3β protein expression was significantly increased in the CCI group compared to sham and CCI + VPA groups. Administration of VPA attenuated CCI-enhanced pGSK-3β proteins. Quantitative fluorescence data are depicted. Data represent the mean ± SEM for 6 rats per group. # p

    Article Snippet: The primary antibodies of GSK-3β, pGSK-3β (1:500 dilution, Cell Signaling Technology, Danvers MA), AKT (1:1000 dilution, Cell Signaling Technology), pAKT (Ser473, 1:500 dilution, Cell Signaling Technology), pNFκB (1:500 dilution, Cell Signaling Technology), COX-2 (1:500 dilution, Abcam London, UK), iNOS (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz CA) and β-actin (1:2000 dilution, Sigma-Aldrich, Saint Louis MO) were used in this experiment.

    Techniques: Staining, Marker, Expressing, Fluorescence

    DARPP-32 regulates EGFR-mediated AKT survival pathway in gastric cancer cells A) Western blot analysis of AKT, pAKT(S473), GSK-3β, and pGSK-3β(S9) in MKN-28 stably expressing DARPP-32 (DP01 and DP02) or empty vector. B) The same as in (A) following transient infection with different titers of adenoviruses expressing DARPP-32 or control. C) The same as in (A) following lentiviral shRNA knockdown of DARPP-32 or control shRNA in SNU-16. D) Human EGFR phosphorylation antibody array using lysates from MKN-28 cells stably expressing DARPP-32 or empty vector, cultured in the presence of gefitinib (25 μM) overnight, demonstrates up-regulation of total and phosphor-EGFR protein in DARPP-32 expressing cells. Quantitative data are shown on the right panel. E–F) Western blot analysis, following overexpression (E) or knockdown of DARPP-32 (F), confirms that results in (D).

    Journal: Gastroenterology

    Article Title: DARPP-32 Increases Interactions Between Epidermal Growth Factor Receptor and ERBB3 to Promote Tumor Resistance to Gefitinib

    doi: 10.1053/j.gastro.2011.06.070

    Figure Lengend Snippet: DARPP-32 regulates EGFR-mediated AKT survival pathway in gastric cancer cells A) Western blot analysis of AKT, pAKT(S473), GSK-3β, and pGSK-3β(S9) in MKN-28 stably expressing DARPP-32 (DP01 and DP02) or empty vector. B) The same as in (A) following transient infection with different titers of adenoviruses expressing DARPP-32 or control. C) The same as in (A) following lentiviral shRNA knockdown of DARPP-32 or control shRNA in SNU-16. D) Human EGFR phosphorylation antibody array using lysates from MKN-28 cells stably expressing DARPP-32 or empty vector, cultured in the presence of gefitinib (25 μM) overnight, demonstrates up-regulation of total and phosphor-EGFR protein in DARPP-32 expressing cells. Quantitative data are shown on the right panel. E–F) Western blot analysis, following overexpression (E) or knockdown of DARPP-32 (F), confirms that results in (D).

    Article Snippet: Horseradish peroxidase-conjugated mouse and rabbit secondary antibodies; ERBB3, EGFR, pEGFR(Y845), AKT, pAKT (S473), GSK-3β, pGSK-3β (S9), and β-actin antibodies were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Infection, shRNA, Ab Array, Cell Culture, Over Expression

    Colocalization of tau phosphorylated at threonine 175 (pThr175 tau) and tau phosphorylated at threonine 231 (pThr 231 tau) Hippocampal neurons in chronic traumatic encephalopathy (CTE) (upper panels) demonstrate colocalization of pThr 175 tau and pThr 231 tau. The presence of pathologic tau oligomers (T22 immunoreactivity) was colocalized to pThr 231 tau-immunoreactive neurons in CTE (middle panels). Consistent with a role in activation of glycogen synthase kinase–3β (GSK3β) in inducing pathologic tau deposition, we observed the colocalization of pThr 175 tau with the active pGSK3β immunoreactivity (lower panel). Tissues were immunolabeled with rabbit anti-pThr 175 tau, rabbit anti-pThr 231 tau, rabbit anti-T22 antibody, and mouse anti-GSK3β pTyr 216 antibody. For double-labeled tissue, red channel antibodies were labeled directly with Alexa Fluor 555. Scale bar = 5 μm.

    Journal: Neurology

    Article Title: Pathologic Thr175 tau phosphorylation in CTE and CTE with ALS

    doi: 10.1212/WNL.0000000000004899

    Figure Lengend Snippet: Colocalization of tau phosphorylated at threonine 175 (pThr175 tau) and tau phosphorylated at threonine 231 (pThr 231 tau) Hippocampal neurons in chronic traumatic encephalopathy (CTE) (upper panels) demonstrate colocalization of pThr 175 tau and pThr 231 tau. The presence of pathologic tau oligomers (T22 immunoreactivity) was colocalized to pThr 231 tau-immunoreactive neurons in CTE (middle panels). Consistent with a role in activation of glycogen synthase kinase–3β (GSK3β) in inducing pathologic tau deposition, we observed the colocalization of pThr 175 tau with the active pGSK3β immunoreactivity (lower panel). Tissues were immunolabeled with rabbit anti-pThr 175 tau, rabbit anti-pThr 231 tau, rabbit anti-T22 antibody, and mouse anti-GSK3β pTyr 216 antibody. For double-labeled tissue, red channel antibodies were labeled directly with Alexa Fluor 555. Scale bar = 5 μm.

    Article Snippet: For colocalizations with pGSK3β, staining was performed with mouse anti-pTyr216 GSK3β antibody (BD Biosciences) followed by secondary labeling with goat anti-mouse Alexa Fluor 633 nm (Invitrogen, Carlsbad, CA).

    Techniques: Activation Assay, Immunolabeling, Labeling

    The effects of neonatal LPS on Nrf2 regulating kinases AKT and GSK-3β and tyrosine hydroxylase (TH) and in substantia nigra of PND70 rats and the effects of a Spirulina enriched diet. Densitometric analyses of phosphoAKT (pAKT), phospho-GSK3β (pGSK3β) and TH protein expression are shown in Fig.4 A, C, E. Data are plotted as ratio of the pAKT/total AKT (Fig. 4A), pGSK3β/total GSK3β (Fig. 4C) and TH/tubulin (Fig. 4E). Representative blots are shown in Fig. 4 B, D, F; respectively. Two-way ANOVA showed a statistically significant interaction between diet and LPS treatment for: phospho-Akt in Fig. 4A, p= 0.004 and for phospho-GSK3β in Fig. 4C, p=0.0320; however the two-way ANOVA did not show statistically significant interaction between diet and treatment for TH. Bonferroni post-hoc analysis comparing Saline (n = 10) vs LPS (n = 9) and LPS vs LPS+SP0.1% (n = 12) groups are shown, *denotes statistical significance (*p

    Journal: Neuroimmunomodulation

    Article Title: Sustained effects of neonatal systemic lipopolysaccharide on IL-1β and Nrf2 in adult rat substantia nigra are partly normalized by a Spirulina enriched diet

    doi: 10.1159/000452714

    Figure Lengend Snippet: The effects of neonatal LPS on Nrf2 regulating kinases AKT and GSK-3β and tyrosine hydroxylase (TH) and in substantia nigra of PND70 rats and the effects of a Spirulina enriched diet. Densitometric analyses of phosphoAKT (pAKT), phospho-GSK3β (pGSK3β) and TH protein expression are shown in Fig.4 A, C, E. Data are plotted as ratio of the pAKT/total AKT (Fig. 4A), pGSK3β/total GSK3β (Fig. 4C) and TH/tubulin (Fig. 4E). Representative blots are shown in Fig. 4 B, D, F; respectively. Two-way ANOVA showed a statistically significant interaction between diet and LPS treatment for: phospho-Akt in Fig. 4A, p= 0.004 and for phospho-GSK3β in Fig. 4C, p=0.0320; however the two-way ANOVA did not show statistically significant interaction between diet and treatment for TH. Bonferroni post-hoc analysis comparing Saline (n = 10) vs LPS (n = 9) and LPS vs LPS+SP0.1% (n = 12) groups are shown, *denotes statistical significance (*p

    Article Snippet: Anti-AKT and anti-phosphorylated AKT antibody (1:1000), Anti-GSK3β (1:500), anti-pGSK3β (1:500) was from Cell Signaling Technology (Massachusetts, USA).

    Techniques: Expressing

    BCR affinity promotes BAFF induced PI3K signaling. ( a,b ) HEL TG B cells were left unactivated (Medium), or stimulated with 100 ng/ml BAFF alone (BAFF only) or in combination with 100 ng/ml HEL, HEL2x or HEL3x in the presence or absence of ( a ) the Pan-PI3K inhibitor Ly249002 or ( b ) the PI3Kδ inhibitor CAL-101. After 48 h viability and Mcl-1 protein levels were assessed by flow cytometry (n = 3). ( c ) HEL TG B cells were stimulated with 100 ng/ml BAFF in combination with 100 ng/ml HEL, HEL2x or HEL3x. After 48 h, cells were deprived from stimuli for 3 h, followed by stimulation with 500 ng/ml BAFF. pAkt T308 levels were measured by intracellular flow cytometry at the indicated time points (n = 3). ( d ) HEL TG B cells were activated with the indicated stimuli. Mcl-1 and pGSK3β levels were determined by western blot. β-actin was used as a loading control. Quantification shows fold induction over the signal on day 0, normalized for β-actin. ( e ) HEL TG B cells were left inactivated (Medium), or stimulated with 100 ng/ml BAFF alone (BAFF only) or in combination with 100 ng/ml HEL, HEL2x or HEL3x in the presence of solvent only (DMSO)or the GSK3 inhibitors XXVI or CHIR99021 (CHIR). All cells were cultured in the presence of 1 μM QVD to exclude caspase-mediated Mcl-1 breakdown. After 24 h Mcl-1 expression was assessed by flow cytometry (n = 3). Values show means ± sem. GMI = Geometric Mean Intensity *P

    Journal: Scientific Reports

    Article Title: Antigen-affinity controls pre-germinal centser B cell selection by promoting Mcl-1 induction through BAFF receptor signaling

    doi: 10.1038/srep35673

    Figure Lengend Snippet: BCR affinity promotes BAFF induced PI3K signaling. ( a,b ) HEL TG B cells were left unactivated (Medium), or stimulated with 100 ng/ml BAFF alone (BAFF only) or in combination with 100 ng/ml HEL, HEL2x or HEL3x in the presence or absence of ( a ) the Pan-PI3K inhibitor Ly249002 or ( b ) the PI3Kδ inhibitor CAL-101. After 48 h viability and Mcl-1 protein levels were assessed by flow cytometry (n = 3). ( c ) HEL TG B cells were stimulated with 100 ng/ml BAFF in combination with 100 ng/ml HEL, HEL2x or HEL3x. After 48 h, cells were deprived from stimuli for 3 h, followed by stimulation with 500 ng/ml BAFF. pAkt T308 levels were measured by intracellular flow cytometry at the indicated time points (n = 3). ( d ) HEL TG B cells were activated with the indicated stimuli. Mcl-1 and pGSK3β levels were determined by western blot. β-actin was used as a loading control. Quantification shows fold induction over the signal on day 0, normalized for β-actin. ( e ) HEL TG B cells were left inactivated (Medium), or stimulated with 100 ng/ml BAFF alone (BAFF only) or in combination with 100 ng/ml HEL, HEL2x or HEL3x in the presence of solvent only (DMSO)or the GSK3 inhibitors XXVI or CHIR99021 (CHIR). All cells were cultured in the presence of 1 μM QVD to exclude caspase-mediated Mcl-1 breakdown. After 24 h Mcl-1 expression was assessed by flow cytometry (n = 3). Values show means ± sem. GMI = Geometric Mean Intensity *P

    Article Snippet: SDS-PAGE gel electrophoresis was performed using the Bio-Rad mini-PROTEAN electrophoresis system with primary antibodies against β-Actin (Santa Cruz Biotechnology), Bim (Stressgen), Bcl-XL (Transduction Laboratories), Bcl-2 (Alexis Biochemicals), Mcl-1 (Rockland), Bax (BD Pharmingen) and pGSK3β (Santa Cruz).

    Techniques: Flow Cytometry, Cytometry, Western Blot, Cell Culture, Expressing

    Effect of HCV infection on insulin-induced phosphorylation of Akt and GSK3β. Huh7.5.1 cells were seeded in a 24-well plate and subjected to serum-free incubation for 20 h, followed by insulin stimulation for the indicated time (0, 15, 30, and 60 min). Western blotting with indicated antibodies revealed a time-dependent change in the phosphorylation of Akt (a) and GSK3β (b) . Furthermore, Huh7.5.1 cells and HCV-infected cells were stimulated with 100 nM insulin for 30 min or left untreated. Extracted proteins were analyzed with antibodies against pAkt, Akt, pGSK3β, and GSK3β (c) .

    Journal: Virology Journal

    Article Title: Inhibition of IRS-1 by hepatitis C virus infection leads to insulin resistance in a PTEN-dependent manner

    doi: 10.1186/s12985-015-0241-4

    Figure Lengend Snippet: Effect of HCV infection on insulin-induced phosphorylation of Akt and GSK3β. Huh7.5.1 cells were seeded in a 24-well plate and subjected to serum-free incubation for 20 h, followed by insulin stimulation for the indicated time (0, 15, 30, and 60 min). Western blotting with indicated antibodies revealed a time-dependent change in the phosphorylation of Akt (a) and GSK3β (b) . Furthermore, Huh7.5.1 cells and HCV-infected cells were stimulated with 100 nM insulin for 30 min or left untreated. Extracted proteins were analyzed with antibodies against pAkt, Akt, pGSK3β, and GSK3β (c) .

    Article Snippet: Antibodies and reagents Insulin and antibodies against IRS-1, pIRS-1 (Ser307), PTEN, Akt, pAkt (Ser473), GSK3β and pGSK3β (Ser9) were purchased from Beyotime, China.

    Techniques: Infection, Incubation, Western Blot

    Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. A: Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. HCT116 cells were transfected with dnIκB, dnAKT or dnTCF4 and were treated with IL-1β or TNFα, or were cultured with THP1 macrophages as indicated. The levels of Snail and c-jun were determined by immunoblotting. B: The levels of Snail and pGSK3β were determined by immunoblotting in Hke-3 cells that were treated with IL-1β, TNFα or were co-cultured with macrophages in the absence or the presence of LY 294002 for 24 hours. C : HCT116 and Hke-3 cells were treated with TRAIL in the absence or the presence of IL-1β and LY294002, as indicated. The experiment was performed three times.

    Journal: PLoS ONE

    Article Title: Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1?- Dependent Stabilization of Snail in Tumor Cells

    doi: 10.1371/journal.pone.0011700

    Figure Lengend Snippet: Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. A: Macrophages, IL-1β and TNFα stabilize Snail in a NF-κB/AKT/Wnt dependent manner. HCT116 cells were transfected with dnIκB, dnAKT or dnTCF4 and were treated with IL-1β or TNFα, or were cultured with THP1 macrophages as indicated. The levels of Snail and c-jun were determined by immunoblotting. B: The levels of Snail and pGSK3β were determined by immunoblotting in Hke-3 cells that were treated with IL-1β, TNFα or were co-cultured with macrophages in the absence or the presence of LY 294002 for 24 hours. C : HCT116 and Hke-3 cells were treated with TRAIL in the absence or the presence of IL-1β and LY294002, as indicated. The experiment was performed three times.

    Article Snippet: Membranes were blocked with 5% milk in TBS containing 0.1% Tween 20, and incubated with antibodies specific for caspase 8 (Abcam), caspase 9, PARP, Snail (Cell Signaling Technology), pGSK3β (Millipore, Billerica, MA), total β-catenin (BD Biosciences, San Jose, CA), and β-actin (Sigma Aldrich, St. Louis, MO).

    Techniques: Transfection, Cell Culture

    MiR-371b-5p utilizes PTEN to orchestrate the PI3K/Akt signaling in hiPSC-ATIICs. a The predicted RNA duplexes of miR-371b-5p target site in the 3′UTR of PTEN ( top panel ). The underlined nucleotides ( UUUGA ) in the target site ( top panel ) were replaced with GGGUC in the mutant 3′UTR construct ( bottom panel ). b Schematic structure of dual-luciferase miRNA target reporter, in which wt or mutant PTEN 3′UTR was inserted into the Pmel and XbaI sites downstream of firefly luciferase (Luc2). c Normalized firefly luciferase activity using the dual-luciferase miRNA target reporter with a wt or mutant PTEN 3′UTR in hiPSC-ATIICs co-transfected with miR-371b-5p mimic. d QRT-PCR analysis of mRNA expression of PTEN in hiPSC-ATIICs transfected with various doses of miR-371b-5p mimic as indicated. The expression levels were normalized to 18 s and presented as percentages of control ( left panel ). PTEN protein expression of each group of miR-371b-5p mimic-transfected hiPSC-ATIICs was analyzed by Western blot using GAPDH as an internal control ( right panel ). e The miR-371b-5p mimic-transfected hiPSC-ATIICs were incubated for 24 hours with and without Rosiglitazone (20 μM). The protein extracts were prepared for Western blot analysis of PTEN, Akt/pAkt, GSK3β/pGSK3β, FOXO3/pFOXO3, and FOXO1/pFOXO1 with GAPDH as control ( left panel ), and the number of viable cells in the cultures was counted 2 days after transfection ( right panel ). ATIICs alveolar epithelial type II cells

    Journal: Stem Cell Research & Therapy

    Article Title: Exosome miR-371b-5p promotes proliferation of lung alveolar progenitor type II cells by using PTEN to orchestrate the PI3K/Akt signaling

    doi: 10.1186/s13287-017-0586-2

    Figure Lengend Snippet: MiR-371b-5p utilizes PTEN to orchestrate the PI3K/Akt signaling in hiPSC-ATIICs. a The predicted RNA duplexes of miR-371b-5p target site in the 3′UTR of PTEN ( top panel ). The underlined nucleotides ( UUUGA ) in the target site ( top panel ) were replaced with GGGUC in the mutant 3′UTR construct ( bottom panel ). b Schematic structure of dual-luciferase miRNA target reporter, in which wt or mutant PTEN 3′UTR was inserted into the Pmel and XbaI sites downstream of firefly luciferase (Luc2). c Normalized firefly luciferase activity using the dual-luciferase miRNA target reporter with a wt or mutant PTEN 3′UTR in hiPSC-ATIICs co-transfected with miR-371b-5p mimic. d QRT-PCR analysis of mRNA expression of PTEN in hiPSC-ATIICs transfected with various doses of miR-371b-5p mimic as indicated. The expression levels were normalized to 18 s and presented as percentages of control ( left panel ). PTEN protein expression of each group of miR-371b-5p mimic-transfected hiPSC-ATIICs was analyzed by Western blot using GAPDH as an internal control ( right panel ). e The miR-371b-5p mimic-transfected hiPSC-ATIICs were incubated for 24 hours with and without Rosiglitazone (20 μM). The protein extracts were prepared for Western blot analysis of PTEN, Akt/pAkt, GSK3β/pGSK3β, FOXO3/pFOXO3, and FOXO1/pFOXO1 with GAPDH as control ( left panel ), and the number of viable cells in the cultures was counted 2 days after transfection ( right panel ). ATIICs alveolar epithelial type II cells

    Article Snippet: Rabbit anti-human Akt/pAkt, Erk/pErk, GSK3β/pGSK3β, FOXO3/pFOXO3, FOXO1/pFOXO1, PTEN, and GAPDH (Cell Signaling, Danvers, MA, USA) were used in the study.

    Techniques: Mutagenesis, Construct, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Incubation

    MiR-371b-5p activates PI3K/Akt pathway in hiPSC-ATIICs. The cultured hiPSC-ATIICs were transfected with various doses of miR-371b-5p mimic as indicated. a Histogram representation of the number of viable cells in the cultures of miR-371b-5p mimic-treated hiPSC-ATIICs. b The miR-371b-5p mimic-transfected hiPSC-ATIICs were treated with BrdU for 12 hours to label the proliferating cells and then were immunostained with mouse anti-BrdU ( red ) and rabbit anti-SPC antibody ( green ). The BrdU-stained images were merged with SPC-stained images ( left panel ), and the BrdU + cells were counted and presented as the percentage of total cells in the miR-371b-5p mimic-treated cultures ( right panel ). c and d Western blot analysis of expression of Akt/pAkt and Erk/pErk in miR-371b-5p mimic-transfected hiPSC-ATIICs ( top panel ). The pAkt and pErk expression levels were quantified and presented as folds of control after being normalized to Akt and Erk, respectively ( bottom panel ). e Western blotting of Akt/pAkt, GSK3β/pGSK3β, FOXO3/pFOXO3, and FOXO1/pFOXO1 of hiPSC-ATIICs treated with miR-371b-5p mimic (150 pmol) using GAPDH as internal control. f The miR-371b-5p mimic-transfected hiPSC-ATIICs were incubated for 24 hours with and without LY294002 (25 μM). The number of viable cells in the cultures was counted 2 days after transfection. ATIICs alveolar epithelial type II cells, BrdU bromodeoxyuridine, DAPI 4′,6-diamidino-2-phenylindole, SPC surfactant protein C

    Journal: Stem Cell Research & Therapy

    Article Title: Exosome miR-371b-5p promotes proliferation of lung alveolar progenitor type II cells by using PTEN to orchestrate the PI3K/Akt signaling

    doi: 10.1186/s13287-017-0586-2

    Figure Lengend Snippet: MiR-371b-5p activates PI3K/Akt pathway in hiPSC-ATIICs. The cultured hiPSC-ATIICs were transfected with various doses of miR-371b-5p mimic as indicated. a Histogram representation of the number of viable cells in the cultures of miR-371b-5p mimic-treated hiPSC-ATIICs. b The miR-371b-5p mimic-transfected hiPSC-ATIICs were treated with BrdU for 12 hours to label the proliferating cells and then were immunostained with mouse anti-BrdU ( red ) and rabbit anti-SPC antibody ( green ). The BrdU-stained images were merged with SPC-stained images ( left panel ), and the BrdU + cells were counted and presented as the percentage of total cells in the miR-371b-5p mimic-treated cultures ( right panel ). c and d Western blot analysis of expression of Akt/pAkt and Erk/pErk in miR-371b-5p mimic-transfected hiPSC-ATIICs ( top panel ). The pAkt and pErk expression levels were quantified and presented as folds of control after being normalized to Akt and Erk, respectively ( bottom panel ). e Western blotting of Akt/pAkt, GSK3β/pGSK3β, FOXO3/pFOXO3, and FOXO1/pFOXO1 of hiPSC-ATIICs treated with miR-371b-5p mimic (150 pmol) using GAPDH as internal control. f The miR-371b-5p mimic-transfected hiPSC-ATIICs were incubated for 24 hours with and without LY294002 (25 μM). The number of viable cells in the cultures was counted 2 days after transfection. ATIICs alveolar epithelial type II cells, BrdU bromodeoxyuridine, DAPI 4′,6-diamidino-2-phenylindole, SPC surfactant protein C

    Article Snippet: Rabbit anti-human Akt/pAkt, Erk/pErk, GSK3β/pGSK3β, FOXO3/pFOXO3, FOXO1/pFOXO1, PTEN, and GAPDH (Cell Signaling, Danvers, MA, USA) were used in the study.

    Techniques: Cell Culture, Transfection, Staining, Western Blot, Expressing, Incubation

    The functional properties of FAK/Wnt signaling pathways in the maintenance of the biological characteristics in the LSC cultured in ESC-CM system. (A) The expression levels of pFAK, FAK, pAkt, Akt, pGSK3β, GSK3β and p21 in the ESC-CM, ESC-CM+FAK inhibitor14 ESC-CM+GSK3β inhibitor or ESC-CM+SiRNA-TERT group. (B) The expression level of β-catenin of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. (C) MTT proliferation assay of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. Data are expressed as the mean±SD (n = 3;*,p

    Journal: PLoS ONE

    Article Title: ES Micro-Environment Enhances Stemness and Inhibits Apoptosis in Human Limbal Stem Cells via the Maintenance of Telomerase Activity

    doi: 10.1371/journal.pone.0053576

    Figure Lengend Snippet: The functional properties of FAK/Wnt signaling pathways in the maintenance of the biological characteristics in the LSC cultured in ESC-CM system. (A) The expression levels of pFAK, FAK, pAkt, Akt, pGSK3β, GSK3β and p21 in the ESC-CM, ESC-CM+FAK inhibitor14 ESC-CM+GSK3β inhibitor or ESC-CM+SiRNA-TERT group. (B) The expression level of β-catenin of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. (C) MTT proliferation assay of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. Data are expressed as the mean±SD (n = 3;*,p

    Article Snippet: The primary antibodies used were mouse anti-p63 mAb, mouse anti-ABCG2 mAb, rabbit anti-FAK, rabbit anti-p-FAK (Try397), rabbit anti-Akt and rabbit anti-p-Akt (Ser473), rabbit anti-GSK3β, rabbit anti-pGSK3β (Try216) and rabbit anti-p21 (all at 1∶1000, Cell Signaling Technology).

    Techniques: Functional Assay, Cell Culture, Expressing, MTT Assay, Proliferation Assay