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: 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: PLoS ONE

Article Title: Augmented Cardiac Hypertrophy in Response to Pressure Overload in Mice Lacking ELTD1

doi: 10.1371/journal.pone.0035779

Figure Lengend Snippet: A, Western blots of MEK1/2, ERK1/2, p38, and JNK phosphorylation and their total protein levels 28 days after surgery in WT and KO mice. GAPDH was used as the sample loading control. (n = 6). Up, representative blots. Down, quantitative results. B, Western blots of Akt,GSK3β, and mTOR phosphorylation and their total protein levels 28 days after surgery. GAPDH was used as the sample loading control (n = 6). Up, representative blots. Down, quantitative results. For A and B, Data represent as the means ± SEM *P<0.01 vs. corresponding sham; #P<0.01 vs. WT/AB after AB.

Article Snippet: Antibodies against the following proteins were purchased from Cell Signaling Technology: p-MEK1/2 (#9154), T-MEK1/2 (#9122), p-ERK1/2 (#4370), T-ERK1/2 (#4695), p-p38 (#4511), T-p38 (#9212), p-JNK1/2 (#4668), T-JNK 1/2(#9258), p-Akt (#4060), T-Akt (#4691), p-GSK3β (#9322), T-GSK3β (#9315), p-mTOR (#2974), T-mTOR (#2972), and GAPDH (#2118).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Never in Mitosis Gene A Related Kinase-6 Attenuates Pressure Overload-Induced Activation of the Protein Kinase B Pathway and Cardiac Hypertrophy

doi: 10.1371/journal.pone.0096095

Figure Lengend Snippet: A and B, The phosphorylated and total protein levels of Akt, GSK-3β, mTOR, eIF4E, and 4E-BP1 in WT and Nek6 KO mice 4 weeks after TAC or sham surgery. Nek6 deficiency augments the activation of Akt signaling. A, representative western blots (duplicate lanes represent two different heart samples); B, quantitative results. * P <0.05 vs . WT/sham; # P <0.05 vs . WT/TAC. C and D, phosphorylated and total protein expression levels of Akt, GSK-3β, mTOR, eIF4E, and 4E-BP1 in H9c2 cells treated with Ang II for the indicated times with or without (control) the overexpression of Nek6. Nek6 overexpression attenuated the activation of Akt signaling. C, representative western blots; D, quantitative results. * P <0.05 vs. the control group at the 0 time point; # P <0.05 vs . the control group at the same time point.

Article Snippet: The primary antibodies against phosphor (P)-Akt Thr308 (2965), total (T)-Akt (4691), P -GSK-3β Ser9 (9322), T-GSK-3β (9315), P-mTOR Ser2448 (2971), T-mTOR (2983), P-4EBP1 (2855p), T-4EBP1 (9644p), P-eIF4e (9741), T-eIF4e (2067) and GAPDH (2118) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Expressing, Over Expression

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Total Saponins of Radix Clematis Regulate Fibroblast-Like Synoviocyte Proliferation in Rheumatoid Arthritis via the LncRNA OIP5-AS1/MiR-410-3p/Wnt7b Signaling Pathway

doi: 10.1155/2022/8393949

Figure Lengend Snippet: Effect of TSC on the expression of GSK-3 β /p-GSK-3 β protein and mRNA in FLS of AA rats. (a) The change in GSK-3 β /p-GSK-3 β protein expression was observed by immunofluorescence analysis (×200): (A) normal group, (B) model group, (C) TSC group, (D) TSC + lncRNA OIP5-AS1 siRNA group, (E) lncRNA OIP5-AS1 siRNA group, and (F) lncRNA OIP5-AS1 siRNA + NC group. (b) The change in GSK-3 β /p-GSK-3 β mRNA expression was observed by RT-qPCR. (c) The change in GSK-3 β /p-GSK-3 β protein expression was observed by Western blotting. (d) Semiquantitative analysis of GSK-3 β /p-GSK-3 β protein expression. ## P < 0.01 compared with the normal group; ∗ P < 0.05 and ∗∗ P < 0.01 compared with the model group.

Article Snippet: Diluted primary antibody: Wnt7b (Abcam, GR3241134-2 1 : 500); β -catenin (Proteintech, 00077341, 1 : 200); c-Myc (Proteintech, 00033258, 1 : 50); cyclin D1 (Abcam, GR197045-1, 1 : 50), p-GSK-3 β (Ser9) (CST, #5588, 1 : 400); SFRP4 (Wuhan Aibotek Biotechnology Co., Ltd., 9100014210, 1 : 500) was added, and samples were placed in a humid box and incubated overnight at 4°C in the dark.

Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: IRS-2/Akt/GSK-3 β /Nrf2 Pathway Contributes to the Protective Effects of Chikusetsu Saponin IVa against Lipotoxicity

doi: 10.1155/2021/8832318

Figure Lengend Snippet: CHS induced the activation of IRS-2/Akt/GSK-3 β signaling in PA-treated β TC3 cells. After being treated with CHS (24 h) and/or PA (another 24 h), the phosphorylation levels of IRS-2 (a), Akt (b), and GSK-3 β (c) were examined. The columns and errors bars are presented as means ± SEM ( n ≥ 5). ## P < 0.01 compared with the CON group. ∗∗ P < 0.01 compared with the PA group. △△ P < 0.01 and △ P < 0.05 compared with the CHS (20 μ M) group.

Article Snippet: Rabbit anti-Nrf2 (#12721, 1 : 1000, Cell Signaling Technology), rabbit anti-IRS-2 (#3089, 1 : 1000, Cell Signaling Technology), rabbit anti-Akt (#9272, 1 : 1000, Cell Signaling Technology), rabbit anti-P-Akt (#4060, 1 : 2000, Cell Signaling Technology), rabbit anti-GSK-3 β (#12456, 1 : 1000, Cell Signaling Technology), rabbit anti-P-GSK-3 β (Ser9) (#5558, 1 : 1000, Cell Signaling Technology), rabbit anti-PDX-1 (#5679, 1 : 1000, Cell Signaling Technology), rabbit anti-SOD1 (#2770, 1 : 1000, Cell Signaling Technology), rabbit anti- β -actin (#4970, 1 : 1000, Cell Signaling Technology), and rabbit anti-Lamin B (#12255, 1 : 1000, Cell Signaling Technology).

Techniques: Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: IRS-2/Akt/GSK-3 β /Nrf2 Pathway Contributes to the Protective Effects of Chikusetsu Saponin IVa against Lipotoxicity

doi: 10.1155/2021/8832318

Figure Lengend Snippet: CHS protected against oxidative stress of pancreatic β TC3 cells from lipotoxicity through GSK-3 β and the relationships between Nrf2 and GSK-3 β . β TC3 cells were treated with CHS in the presence or absence of LiCl (GSK-3 β inhibitor) for 24 h and then subjected to PA for another 24 h. Effects of LiCl (GSK-3 β inhibitor) on CHS-induced GSK-3 β phosphorylation and PDX-1 expression levels were measured (a). Immunoblot analysis showed the protein expression of Nrf2 in β TC3 cells pretreated with CHS (24 h) with or without PA (24 h) (b). Effects of LiCl on Nrf2 transcriptional activity (c), ROS (d), SOD (e), cell viability (f), and insulin levels (g) were measured after different treatments. The columns and errors bars are presented as means ± SEM ( n ≥ 5). ## P < 0.01 compared with the CON group, ∗∗ P < 0.01 compared with the PA group, and && P < 0.01 compared with the CHS treatment group. (h) Potential mechanism underlying the protective effects of CHS on lipotoxicity-induced cell injuries.

Article Snippet: Rabbit anti-Nrf2 (#12721, 1 : 1000, Cell Signaling Technology), rabbit anti-IRS-2 (#3089, 1 : 1000, Cell Signaling Technology), rabbit anti-Akt (#9272, 1 : 1000, Cell Signaling Technology), rabbit anti-P-Akt (#4060, 1 : 2000, Cell Signaling Technology), rabbit anti-GSK-3 β (#12456, 1 : 1000, Cell Signaling Technology), rabbit anti-P-GSK-3 β (Ser9) (#5558, 1 : 1000, Cell Signaling Technology), rabbit anti-PDX-1 (#5679, 1 : 1000, Cell Signaling Technology), rabbit anti-SOD1 (#2770, 1 : 1000, Cell Signaling Technology), rabbit anti- β -actin (#4970, 1 : 1000, Cell Signaling Technology), and rabbit anti-Lamin B (#12255, 1 : 1000, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Activity Assay