Journal: The Journal of Biological Chemistry

Article Title: Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat

doi: 10.1016/j.jbc.2023.103059

Figure Lengend Snippet: Acute diosmin (Dios) administration improves diabetic gene programs in iWAT of mice. A , experimental model of acute control (Con) or Dios administration in mice with iWAT unilateral injection (n = 4). B , protein levels of S273 p-PPARγ, ( C ) p-IRβ, p-AKT, and p-GSK3β, ( D ) expression of gene set regulated by PPARγ S273 phosphorylation in iWAT of mice after acute Dios administration. Data are presented as mean ± SEM and ∗ p < 0.05, ∗∗ p < 0.01 compared with control group. iWAT, inguinal white adipose tissue; PPARγ, peroxisome proliferator–activated receptor γ.

Article Snippet: Membranes were incubated in 5% bovine serum albumin for 2 h and with primary antibodies overnight at 4 °C, including anti-p-PPARγ (1:2000 dilution) (catalog no.: bs-4888R; Bioss biotech), anti-PPARγ (1:1000 dilution) (catalog no.: sc-7273; Santa Cruz), anti-p-IRβ (1:2000 dilution) (catalog no.: 3025; Cell Signaling Technology), anti-p-AKT (1:2000 dilution) (catalog no.: 13038; Cell Signaling Technology), anti-p-GSK3β (1:2000 dilution) (catalog no.: 9322; Cell Signaling Technology) (1:2000 dilution), anti-UCP1 (catalog no.: Ab10983; Abcam), or β-actin (1:2000 dilution) (catalog no.: sc-47778; Santa Biotechnology).

Techniques: Injection, Expressing

Journal: Cancers

Article Title: P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

doi: 10.3390/cancers15030877

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Phospho GSK-ß 9322 , 1:1000 , Cell Signaling , LC3 ab51520 , 1:3000 , Abcam.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: APPsα restores normal GSK3β activity and modulates the Akt/GSK3β pathway in THY-Tau22 mice. (A) Schematic overview of the regulation of GSK3β activity. Activated Akt (pAkt Ser473 ) negatively regulates the activity of GSK3β through phosphorylation of Ser 9 , which leads to GSK3β inactivation. (B) Western blot analysis of hippocampi from AAV-Venus or AAV-APPsα injected littermates (LM) or THY-Tau22 mice. Specific antibodies were used to detect total GSK3β and inactive pGSK3β Ser9 . Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (C) No differences were detected for total GSK3β between groups. (D) THY-Tau22-Venus mice revealed a strong trend toward reduced GSK3β activity, as shown by signal intensities of inactive pGSK3β Ser9 normalized to that of total GSK3β (LM-Venus vs. THY-Tau22-Venus, p = 0.060). AAV-APPsα expression restored GSK3β activity to littermate control level (THY-Tau22-Venus vs. THY-Tau22-APPsα, p = 0.051). (E) Radioactive kinase assay involving Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of GSK3β from AAV-Venus or AAV-APPsα injected littermates and THY-Tau22 mice. Radioactively labeled Tau was visualized using PI. Recombinant Tau (HT7), GSK3β and CDK5 were visualized by immunodetection using specific monoclonal antibodies. Note the absence of CDK5 after immunoprecipitation of GSK3β. (F) Quantitative analysis revealed significantly increased GSK3β activity (PI signal normalized to total immunoprecipitated GSK3β, WB signal) in THY-Tau22-Venus mice compared to LM-Venus mice (LM-Venus vs. THY-Tau22-Venus, ** p = 0.007). AAV-APPsα restored normal GSK3β activity (THY-Tau22-Venus vs. THY-Tau22-APPsα, ** p = 0.007). (G) Western blot analysis of total Akt and active Akt (pAkt Ser473 ) in THY-Tau22 mice after AAV-Venus or AAV-APPsα injection. Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (H,I) Quantitative analysis of the Western blot depicted in (G) . THY-Tau22 mice showed a reduction in (H) the total expression of Akt (LM-Venus vs. THY-Tau22-Venus, ** p = 0.003) and (I) for the activating Ser 473 phosphorylation of Akt (LM-Venus vs. THY-Tau22-Venus, *** p = 0.0005). AAV-APPsα rescued both total Akt and pAkt 473 (THY-Tau22-Venus vs. THY-Tau22-APPsα, *** p = 0.0002 and *** p = 0.0009), respectively. Data are depicted as mean ± SEM; N, number of animals; age of analysis, 12 months, data were analyzed using one-way ANOVA with Tukey post hoc test.

Article Snippet: The following antibodies were used: AT8 (mouse monoclonal, 1:500, #MN1020, Thermo Fisher Scientific), AT180 (mouse monoclonal, 1:1000, #MN1040, Thermo Fisher Scientific), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific), MC1 (mouse monoclonal, 1:500, kindly provided by Peter Davies), α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-p35/p25 (rabbit monoclonal, 1:1000, #2680, Cell Signaling Technology), α-Vinculin (mouse monoclonal, 1:1000, #sc-73614, Santa Cruz Biotechnology), α-GAPDH (rabbit polyclonal, 1:2000, #ABS16, Merck Millipore), α-beta-Tubulin (mouse monoclonal, 1:10000, #MAB3408, Merck Millipore), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), α-pGSK3β Ser9 (rabbit monoclonal, 1:1000, #9322, Cell Signaling Technology), α-GFAP (rabbit polyclonal, 1:1000, #173002, Synaptic Systems), α-HA-tag (mouse monoclonal, 1:1000, #2367, Cell Signaling Technology), α-PSD95 (mouse monoclonal, 1:1000, #MAB1598, Merck Millipore), α-β-catenin (mouse monoclonal, 1:500, #sc-7963, Santa Cruz Biotechnology), α-pβ-catenin Ser33/37/Thr41 (rabbit polyclonal, 1:1000, #9561, Cell Signaling Technology), M3.2 (mouse monoclonal, 1:1000, a kind gift from Paul Mathews), α-Akt (rabbit monoclonal, 1:1500, #4691, Cell Signaling Technology), α-pAkt Ser473 (rabbit monoclonal, 1:1000, #4060, Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Injection, Staining, Expressing, Kinase Assay, Immunoprecipitation, Labeling, Recombinant, Immunodetection

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