Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Experimental scheme for this study. After gene identification at 1 month of age, 3-month-old male WT and 3×Tg-AD mice were randomly assigned to four groups with 10 animals each and then intragastrically administered either ICA or vehicle for 5 months (WT + vehicle, WT + ICA, 3×Tg-AD + vehicle, 3×Tg-AD + ICA groups). After performing behavior tests, the mice were euthanized. The cerebral cortexes were evaluated using HE and Nissl staining, immunofluorescent staining, and western blot assays to determine the above disease indicators. 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; Aβ: beta-amyloid protein; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; HE: hematoxylin and eosin; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; NeuN: neuronal nuclear antigen; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PSD95: postsynaptic density protein 95; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Staining, Western Blot, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Effects of ICA on impaired insulin signaling in the cerebral cortex of 3×Tg-AD mice. (A) Insulin signaling: IR tyrosine autophosphorylation is stimulated by insulin and triggers IRS1 phosphorylation at tyrosine residues, which represents a positive regulatory mechanism that activates the PI3K/AKT pathway and results in the inhibition of GSK3β. However, serine phosphorylation of IRS1 at specific sites is a negative regulatory mechanism. (B) Representative expression patterns of molecules related to the insulin signaling pathway. (C) Quantification of proteins related to the insulin signaling pathway shown in (B). Protein levels were normalized to those in the WT + vehicle group. The data are presented as the means ± SEM ( n = 4–6). * P < 0.05, vs . WT + vehicle group; # P < 0.05, vs . 3×Tg-AD + vehicle group (one-way analysis of variance followed by the least significant difference test). 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; AKT: protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Inhibition, Expressing, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Schematic diagram of the mechanism by which ICA regulates GLUTs and brain insulin signaling to ameliorate memory impairment in AD. Aβ: Amyloid-beta protein; AD: Alzheimer’s disease; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; G-tau: the attachment of O-linked N-acetylglucosamine (O-GlcNAc) on tau; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques:

Journal: Disease Markers

Article Title: Leptin Promotes HTR-8/SVneo Cell Invasion via the Crosstalk between MTA1/WNT and PI3K/AKT Pathways

doi: 10.1155/2022/7052176

Figure Lengend Snippet: Leptin mediates β -catenin activation through the crosstalk between MTA1/WNT and PI3K/AKT pathways in HTR-8/SVneo cells. (a) HTR-8/SVneo cells were treated with exogenous leptin (0 and 200 ng/ml) for 24 h, and MTA1, WNT1, p-GSK3 β (Ser9), and p-AKT (Ser473) levels were detected by Western blot. Data are shown as mean ± SD; ∗ P < 0.01 vs. leptin (0 ng/ml). (b) The knockdown efficiencies of MTA1 and WNT1 were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (c) HTR-8/SVneo cells were transfected with MTA1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of MTA1, WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (d) HTR-8/SVneo cells were transfected with WNT1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (e) The knockdown efficiencies of AKT and β -catenin were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (f) HTR-8/SVneo cells were transfected with AKT siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of AKT, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. All experiments were performed in triplicate.

Article Snippet: The following primary antibodies were used: MMP9 (ab76003), MTA1 (ab71153), WNT1 (ab15251), AKT (ab179463), GSK3 β (ab32391), β -catenin (ab32572), histone 3 (ab1791), β -actin (ab6276) (Abcam, USA); p-AKT (Ser473) (#4060), p-GSK3 β (Ser9) (#9322) (Cell Signaling Technology, USA).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Transfection, Expressing

Journal: Cell Death Discovery

Article Title: Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

doi: 10.1038/s41420-022-01291-z

Figure Lengend Snippet: A , B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C , D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Article Snippet: The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).

Techniques: Activation Assay, Western Blot, In Vitro, In Vivo, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Cell Death Discovery

Article Title: Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

doi: 10.1038/s41420-022-01291-z

Figure Lengend Snippet: A , B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C , D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Article Snippet: The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).

Techniques: Activation Assay, Western Blot, In Vitro, In Vivo, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Effect of repeated intranasal LPS challenge and treatment with the selective GSK-3 inhibitor SB216763 on extracellular matrix deposition in the lung. (A) Expression of fibronectin was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. Effects of repeated LPS challenge and SB216763 treatment on fibronectin expression were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (B) Histological staining of the extracellular matrix protein collagen using Sirius Red. The non-cartilaginous airways were digitally photographed (100-200 × magnification) and analysed by using ImageJ software. Effects of repeated LPS challenge and SB216763 treatment on airway collagen expression were quantified, representing mean ± s.e.m. of 9 animals per group. (C) The mean linear intercept (LMI), a measure for alveolar airspace size, was determined by staining the tissue-sections with haematoxylin and eosin. Data represent means ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Staining, Software

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Repeated LPS instillation and pharmacological inhibition of GSK-3 by SB216763 do not affect airway smooth muscle content. Immunohistological analysis of sm-MHC positive area in (A) large (cartilaginous) and (B) small (non-cartilaginous) airways. Effects of repeated LPS challenge and SB216763 treatment on airway smooth muscle sm-MHC expression were quantified. Data represent means ± s.e.m. of 9 animals per group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Right ventricle hypertrophy after repeated intranasal LPS instillation is attenuated by GSK-3 inhibition. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on right ventricle hypertrophy. Effects of repeated LPS challenge and SB216763 treatment on size of right ventricle were quantified as right ventricle weight over total heart weight, representing mean ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: GSK-3 inhibition does not inhibit LPS-induced pulmonary inflammation. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on inflammatory cell infiltration in the airways. Cells within 50 μm of the basement membrane were quantified and expressed relative to basement membrane length, representing mean ± s.e.m. of 9 animals per group. *p < 0.05 compared to control group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Activation of β-catenin in response to repeated intranasal LPS challenge is prevented by treatment with the selective GSK-3 inhibitor SB216763. (A) Expression of active β-catenin, phosphorylated GSK-3 (ser9/21 GSK-3) and total GSK-3 was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. (B,C) Responses of repeated LPS challenge and SB216763 treatment on active β-catenin expression (B) and GSK-3 phosphorylation (C) were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (D) Correlation between pulmonary expression of fibronectin (data from Figure ) and active β-catenin in all guinea pigs. R = 0.552; p < 0.001. (E) Immunofluorescence analysis of active β-catenin (red) in large airways counterstained with Hoechst 3342 to stain nuclei (blue). *p < 0.05 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining

Journal: BioMed Research International

Article Title: The Effects of Omega-3 Fatty Acid Supplementation on Dexamethasone-Induced Muscle Atrophy

doi: 10.1155/2014/961438

Figure Lengend Snippet: Protein expression in GA muscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last 10 days). Relative optical density of total Akt (tAkt) and phosphorylated Akt (P-Akt) ((a) and (c)), total GSK3 β (tGSK3 β ) and phosphorylated GSK3 β (P-GSK3 β ) ((b) and (d)), and tAkt/P-Akt and tGSK3 β /P-GSK3 β ratios ((e) and (f), resp.). Bands representing tAkt, P-Akt, tGSK3, and P-GSK3 β are illustrated in (g). α -Tubulin (55 kDa) was used as control. The numbers represent the mean ± S.E.M. CT = control group ( n = 6), DX = dexamethasone group ( n = 5), n-3 = n-3 PUFA group ( n = 6), and DX + n-3 = dexamethasone + n-3 PUFA group ( n = 6). Values represent means ± S.E.M., analyzed by Student's t -test, # P < 0.05.

Article Snippet: The primary antibodies used were Akt (1 : 2,000, Cell Signaling #4691), P-Akt (1 : 2,000, Cell Signaling #4060, Ser473); mTOR (1 : 500, Cell Signaling #2972), P-mTOR (1 : 500, Cell Signaling #2971, Ser2448), GSK3 β (1 : 2,000, Cell Signaling #9315), P-GSK3 β (1 : 2,000, Cell Signaling #9322, Ser9), FoXO3a (1 : 500, Cell Signaling #2497), P-FoXO3a (1 : 500, Cell Signaling #9466, Ser253), and α -tubulin (1 : 30,000, Hybridoma bank) in 5% BSA/TBS-T.

Techniques: Expressing

Journal: Stem Cells International

Article Title: Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

doi: 10.1155/2017/3508907

Figure Lengend Snippet: Effect of ASP on Wnt/ β -catenin signaling mediators of Sca-1 + HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) GSK-3 β , Ser9-phosphorylated GSK-3 β and TCF-4 protein expressions by Western blot. β -actin was used as the internal control. (b) The relative protein expression of GSK-3 β . (c) The relative protein expression of Ser9-phosphorylated GSK-3 β . (d) The relative protein expression of TCF-4. Different letters: P < 0.05.

Article Snippet: Membranes were incubated overnight at 4°C with the primary antibodies against β -catenin, GSK-3 β , Phospho-GSK-3 β , TCF-4, γ -H2A.X (all purchased from Cell Signaling Technology, USA, catalog number: 9562, 9832, 9336, 2566, 2577), p16 INK4a , Rb, p53, p21 Cip1/Waf1 , β -actin (all purchased from Santa Crus, USA, catalog number: sc-73434, sc-1538, sc-99, sc-397, sc-130656), and 4-HNE (ab46545, Abcam, USA), respectively.

Techniques: Western Blot, Expressing

Journal: Stem Cells International

Article Title: Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

doi: 10.1155/2017/3508907

Figure Lengend Snippet: Effect of ASP on Wnt/ β -catenin signaling mediators of Sca-1 + HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) GSK-3 β , Ser9-phosphorylated GSK-3 β and TCF-4 protein expressions by Western blot. β -actin was used as the internal control. (b) The relative protein expression of GSK-3 β . (c) The relative protein expression of Ser9-phosphorylated GSK-3 β . (d) The relative protein expression of TCF-4. Different letters: P < 0.05.

Article Snippet: Membranes were incubated overnight at 4°C with the primary antibodies against β -catenin, GSK-3 β , Phospho-GSK-3 β , TCF-4, γ -H2A.X (all purchased from Cell Signaling Technology, USA, catalog number: 9562, 9832, 9336, 2566, 2577), p16 INK4a , Rb, p53, p21 Cip1/Waf1 , β -actin (all purchased from Santa Crus, USA, catalog number: sc-73434, sc-1538, sc-99, sc-397, sc-130656), and 4-HNE (ab46545, Abcam, USA), respectively.

Techniques: Western Blot, Expressing