Journal: iScience

Article Title: Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target

doi: 10.1016/j.isci.2023.107047

Figure Lengend Snippet: The combined effect of spaceflight across all missions on GSK3 content, phosphorylation status and MHC isoform content in soleus muscles (A) Total GSK3β content. (B) Phosphorylated GSK3β (Ser9) relative to total GSK3β content. (C) Total GSK3α content. (D) Phosphorylated GSK3α (Ser21) relative to total GSK3β content. (E) MHC I content. (F) MHC IIa content. (G) MHC IIx content. (H) MHC IIb content. Fold-change (FC) data were calculated for each mission by dividing the Flight group by the average of the combined GC and VIV groups. The combined dataset represents the mean ± SEM and p value (Student’s t test) for all FC data (RR1, RR9, RR18, and BION-M1).

Article Snippet: Antibodies from pGSK3β (9336), total (t)-GSK3-β (9315), pGSK3α (9316), tGSK3α (4818), β-Catenin (8480) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: iScience

Article Title: Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target

doi: 10.1016/j.isci.2023.107047

Figure Lengend Snippet: Partial muscle-specific Gsk3 knockdown (GSK3 mKD ) increases soleus muscle mass, myogenic signaling, and the oxidative phenotype while preserving muscle strength after 7 days of hindlimb suspension (HLS) (A–C) DXA scan analyses showing that GSK3 mKD mice have no change in body mass but have lowered % fat mass and increased % lean mass even after 7 days of HLS. (D and E) Absolute and relative (to body mass) soleus muscle weights. (F and G) Percent reduction of absolute and relative soleus muscle weights in GSK3 mKD and GSK3 flox mice when compared to their respective mobile controls (see Figure S10 ). (H–J) H&E staining in the soleus shows that GSK3 mKD mice have an increased distribution of larger fibers versus GSK3 flox mice (rightward shift) and increased centrally located nuclei (see yellow arrows). Scale bars are set to 200 μm; CSA, cross-sectional area. (K) Western blot analysis of myogenic markers Pax7 and myogenin. (L) Western blot analysis of oxidative phenotype markers, MHC I, MHC IIa, PGC-1α, and COXIV as well as the glycolytic MHC IIx. (M) Specific force-frequency curves in soleus muscles from GSK3 mKD and GSK3 flox control mice after 7 days of HLS. (N) Specific force-frequency curves in soleus muscles from mobile GSK3 mKD and GSK3 flox control mice. (O) Calculated percent reduction in specific force across stimulation frequencies from GSK3 mKD and GSK3 flox control mice after 7 days of HLS (compared to their respective mobile controls). For (B, C, E, J, K, L), ∗p < 0.05 using a Student’s t test. For (N-O), a two-way ANOVA was used to test the main effects of genotype and frequency. Data are presented as means ± SEM.

Article Snippet: Antibodies from pGSK3β (9336), total (t)-GSK3-β (9315), pGSK3α (9316), tGSK3α (4818), β-Catenin (8480) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Preserving, Staining, Western Blot

Journal: iScience

Article Title: Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target

doi: 10.1016/j.isci.2023.107047

Figure Lengend Snippet: GSK3β phosphorylation and content in femur samples obtained from male RR9 mice (A and B) Bone mineral content (BMC) and bone mineral density (BMD) of the individual bones obtained from a small animal DXA scanner. (C) Representative western blot images of phosphorylated (Ser9) and total GSK3β. (D and E) Western blot analysis of phosphorylated (Ser9) and total GSK3β content normalized to ponceau. (F) GSK3 activation status measured as the ratio of phosphorylated (Ser9) GSK3β relative to total GSK3β. (G) Representative DXA scan showing region-specific analysis of the femur, tibia, and lumbar spine in mice. (H) Region-specific BMD analysis in mobile GSK3 mKD and GSK3 flox mice measured at baseline. (I) Region-specific BMD analysis in mobile GSK3 mKD and GSK3 flox mice measured after 7 days of HLS. (J) Western blot analysis of soleus muscle FNDC5 from GSK3 mKD and GSK3 flox mice measured after 7 days of HLS. (K) Proposed tissue crosstalk between muscle and bone with muscle-specific Gsk3 deletion leading to an increase in FNDC5 and tibia BMD. ∗p < 0.05, ∗∗∗p < 0.001 using a Student’s t test (n = 6–12 per group for (A–F); n = 3–5 per group for (H–J). For (A–F), GC and VIV controls were combined to increase statistical power. All values are presented as means ± SEM.

Article Snippet: Antibodies from pGSK3β (9336), total (t)-GSK3-β (9315), pGSK3α (9316), tGSK3α (4818), β-Catenin (8480) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Activation Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Multifunctional Effects of Mangosteen Pericarp on Cognition in C57BL/6J and Triple Transgenic Alzheimer's Mice

doi: 10.1155/2014/813672

Figure Lengend Snippet: List of the primary antibodies used in this study.

Article Snippet: GSK3 α , Rabbit , Cell Signaling , 1 : 1,000 , — , Total GSK3 α.

Techniques: Binding Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Zi Shen Decoction Inhibits Growth and Metastasis of Lung Cancer via Regulating the AKT/GSK-3 β / β -Catenin Pathway

doi: 10.1155/2021/6685282

Figure Lengend Snippet: ZSD induced the apoptosis of H1299 cells partially by regulating AKT/GSK-3 β / β -catenin signaling. (a) ZSD induced the apoptosis of H1299 cells after treatment with ZSD (4 mg/mL) for 12 h, 24 h, and 48 h. (b) ZSD induced apoptosis of H1299 cells in a dose-dependent manner. (c) Pathway enrichment analysis of the genes in Human Signal Transduction Pathway Finder PCR Array. (d) The mRNA expression of AKT/GSK-3 β / β -catenin signal cascades in H1299 cells after treatment with ZSD (4 mg/mL). ∗ p < 0.05 and ∗∗ p < 0.01 vs. the control group.

Article Snippet: Antibodies against total AKT and p-AKT, total GSK-3 β and p-GSK-3 β , β -catenin, cleaved caspase-3, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transduction, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Zi Shen Decoction Inhibits Growth and Metastasis of Lung Cancer via Regulating the AKT/GSK-3 β / β -Catenin Pathway

doi: 10.1155/2021/6685282

Figure Lengend Snippet: ZSD inhibited the AKT/GSK-3 β / β -catenin pathway in lung cancer cells and tumor tissues. (a) Western blot analysis was used to assess the expression of multiple proteins involved in the AKT/GSK-3 β / β -catenin pathway in lung cancer cells after ZSD treatment for 12 h, 24 h, and 48 h. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. the control group. (b) Western blots showing the protein expression of AKT, p-AKT, GSK-3 β , p-GSK-3 β , β -catenin, cleaved caspase-3, and Bax and Bcl-2 in isolated tumor tissues. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. the model group.

Article Snippet: Antibodies against total AKT and p-AKT, total GSK-3 β and p-GSK-3 β , β -catenin, cleaved caspase-3, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Isolation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Zi Shen Decoction Inhibits Growth and Metastasis of Lung Cancer via Regulating the AKT/GSK-3 β / β -Catenin Pathway

doi: 10.1155/2021/6685282

Figure Lengend Snippet: The AKT/GSK-3 β / β -catenin pathway medicated anticancer effect of ZSD in lung cancer cells. H1299 cells were treated with ZSD and/or SC-79 for 24 h. (a) Representative western blots and quantitative analysis of the AKT/GSK-3 β / β -catenin pathway related proteins. (b) The mobility of H1299 cells was detected by wound-healing assay. (c) The migration and invasion abilities of H1299 cells were detected by transwell assays. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. the control group.

Article Snippet: Antibodies against total AKT and p-AKT, total GSK-3 β and p-GSK-3 β , β -catenin, cleaved caspase-3, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Wound Healing Assay, Migration

Journal: PLoS ONE

Article Title: Lithium Improves Hippocampal Neurogenesis, Neuropathology and Cognitive Functions in APP Mutant Mice

doi: 10.1371/journal.pone.0014382

Figure Lengend Snippet: Antibodies employed in the study.

Article Snippet: GSK 3β , Total GSK-3 β protein , 1∶1000 , Not done , Rabbit , Cell Signaling.

Techniques: Binding Assay

Journal: International Journal of Endocrinology

Article Title: Ghrelin Increases Beta-Catenin Level through Protein Kinase A Activation and Regulates OPG Expression in Rat Primary Osteoblasts

doi: 10.1155/2015/547473

Figure Lengend Snippet: Representative Western blot (a) and quantification (b) of timecourse experiments performed on rat osteoblast-like cells treated with ghrelin (AG, 10 −10 M) for 15, 30, 60, and 90 min. Phosphorylated-GSK-3 β (P-GSK-3 β ) levels were measured in whole cell lysates and normalized to total GSK-3 β levels (Tot-GSK3 β ). Results are shown as ratio of P-GSK3 β measured in treated versus untreated cells. Data are the mean ± SEM of 5 experiments * P < 0.05, versus untreated cells.

Article Snippet: Western blots were performed using a specific antibody against rat β -catenin diluted 1 : 5000 in 5% milk in Tris–HCl with 0.1% Tween-20 (TBST); rat P-GSK3 β (1 : 2000 5% milk in TBST); rat total GSK3 β (1 : 2000 5% milk in TBST) (Cell Signaling Technology, Boston, MA) or against rat β -actin (1 : 2000 5% milk in TBST) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Western Blot

Journal: International Journal of Endocrinology

Article Title: Ghrelin Increases Beta-Catenin Level through Protein Kinase A Activation and Regulates OPG Expression in Rat Primary Osteoblasts

doi: 10.1155/2015/547473

Figure Lengend Snippet: Effect of the pretreatment with the PKA inhibitor H89 (2 × 10 −6 M, 30 min before) on (a) β -catenin and (b) phosphorylated-GSK3 β (P-GSK3 β ) levels measured in whole cell lysates by Western blot on rat osteoblast-like cells (rOB) treated with ghrelin (AG, 10 −10 M for 1 h). β -catenin levels were normalized to β -actin levels; P-GSK3 β was normalized to total GSK3 β levels (Tot-GSK3 β ). Results are expressed as ratio of β -catenin or P-GSK3 β measured in treated cells versus untreated cells. Insets : typical gels obtained from rOB cell lysates after treatment with ghrelin alone or together with H89. Data are the mean ± SEM of 5 experiments * P < 0.05 versus untreated cells; § P < 0.05 versus AG treated cells.

Article Snippet: Western blots were performed using a specific antibody against rat β -catenin diluted 1 : 5000 in 5% milk in Tris–HCl with 0.1% Tween-20 (TBST); rat P-GSK3 β (1 : 2000 5% milk in TBST); rat total GSK3 β (1 : 2000 5% milk in TBST) (Cell Signaling Technology, Boston, MA) or against rat β -actin (1 : 2000 5% milk in TBST) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras -Induced Lung Cancer

doi: 10.1371/journal.pone.0002220

Figure Lengend Snippet: (A) PI3K expression increases with malignant progression. Representative immunohistochemical staining of lesions in the tissue microarray and their quantification in adjacent bar graphs. Calibration bar in lower right panel represents 100 μm. Scores of mixed (solid/papillary) adenomas are included in bar graphs but not in PI3K staining images. (B) PI3K is required for tumor growth. Mean changes in tumor multiplicity and volume determined by micro-computed tomography performed before and after treatment. (* P <0.05 compared to vehicle). (C) PX-866 inhibits PI3K in lung tissues. Western blotting of Ser473-phosphorylated AKT (P-AKT) and Ser21/9-phosphorylated GSK3 (P-GSK3α/β) in whole lung lysates from mice (n = 7 in each treatment cohort). Positions of molecular weight markers are indicated on the left. (D) PX-866 decreases tumor cell proliferation. Representative immunohistochemical staining of Ser28-phosphorylated histone H3 (P-H3) in adenomas in the tissue microarray (x 20 magnification, areas with positive cells encircled) and quantification of staining in 16 lesions from vehicle-treated mice and 4 lesions from PX-866-treated mice (bar graph, * P <0.05 compared to vehicle). Inset illustrates encircled cells at ×40 magnification. Calibration bars represent 200 μm.

Article Snippet: Antibodies purchased for these studies include rabbit anti-p110α (sc-7174), p110β (sc-7175), SPC (sc-13979), goat anti-CC10 (sc-9772) and PTEN (sc-6818) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-total AKT (9272), Ser473-phosphorylated AKT (9271), Ser21/9-phosphorylated GSK3α/β (9331), total GSK3 (9332), cleaved caspase-3 (9661), Poly (ADP-ribose) polymerase (9542), phosphorylated-histone H3 (9701), Ser28-phosphorylated histone H3 (9713P) (Cell Signaling Technologies, Danvers, MA), rabbit anti-SPC (WRAB-SPC, Seven Hills Bioreagents, Cincinnati, OH), rabbit anti-CCSP (07-623, Upstate Biotechnologies, Lake Placid, NY), FITC-conjugated anti-Sca-1 (557405), biotin-conjugated anti-CD45 (553771) biotin-conjugated-CD31 (558737), PE-conjugated anti-CD34 (12-0341-33, eBioscience), anti-rabbit IgG Alexa Flour 488 (A11008), and anti-goat IgG Alexa Flour 594 (A11058) (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Micro-CT, Western Blot, Molecular Weight

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats

doi: 10.1155/2013/459467

Figure Lengend Snippet: Western blot analysis of phosphorylation levels of Akt, GSK3 β , and c-Raf at 24 h after reperfusion. (a) Representative protein bands for p-Akt (Ser473), total Akt. p-Akt (Ser473) expression levels significantly were decreased at 24 h after reperfusion, whereas XXMD treatment enhanced p-Akt (Ser473) levels and the effects could be partly reversed by PI3K inhibitor. No changes in Akt were detected in rats of different groups. GAPDH was used to show equal protein loading of each lane. (b) Representative protein bands for p-GSK3 β (Ser9), total GSK3 β . A decrease in p-GSK3 β (Ser9) level was observed in the peripheral area of ischemia after reperfusion and the levels of p-GSK3 β (Ser9) in the XXMD60 group were higher than those in the I/R group. However, inhibition of PI3K using LY294002 abolished the increase. No changes in GSK3 β were observed in rats of different groups. (c) Representative protein bands for p-c-Raf, total c-Raf. Although p-c-Raf expression levels significantly were decreased at 24 h after reperfusion, XXMD preserved the levels of p-c-Raf. Notably, LY294002 did not significantly block the effect of XXMD on p-c-Raf. No changes in c-Raf were detected in rats of different groups. Data are reported as the means ± SEM. n = 5; * P < 0.05 versus the I/R group.

Article Snippet: LY294002 (PI3K inhibitor), the primary antibodies for phospho-PDK1 (p-PDK1, Ser241), total PDK1, phospho-PTEN (p-PTEN, Ser380), total PTEN, phospho-Akt (p-Akt, Ser473), total Akt, phospho-c-Raf (p-c-Raf, Ser259), total c-Raf, phospho-GSK3 β (p-GSK3 β , Ser9), total GSK3 β , GAPDH, and horseradish-peroxidase- (HRP-) linked anti-rabbit antibody purchased from Cell Signaling Technology (Beverly, MA, USA) were used for Western blot analysis.

Techniques: Western Blot, Expressing, Inhibition, Blocking Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats

doi: 10.1155/2013/459467

Figure Lengend Snippet: The diagram of the cell signaling pathway which was involved in NVU protection of XXMD against cerebral injury following focal cerebral ischemia and reperfusion. Reperfusion after stroke induced dysfunction of PI3K/Akt pathway and the PI3K/Akt inhibition led to dephosphorylation of PDK1, Akt, and GSK3 β degradation, resulting in apoptosis. However, XXMD treatment inhibited apoptosis of different cells in NVU and increased expression levels of p-PTEN, p-PDK1, p-Akt, and p-GSK3 β . Furthermore, PI3K/Akt pathway is associated with Ras/MAPK pathway. In the present study, we also found that XXMD upregulated the level of p-c-raf. Above all, all the observations in the study indicated that XXMD may exert its NVU protection partly through the activation of PI3K/Akt signaling pathway.

Article Snippet: LY294002 (PI3K inhibitor), the primary antibodies for phospho-PDK1 (p-PDK1, Ser241), total PDK1, phospho-PTEN (p-PTEN, Ser380), total PTEN, phospho-Akt (p-Akt, Ser473), total Akt, phospho-c-Raf (p-c-Raf, Ser259), total c-Raf, phospho-GSK3 β (p-GSK3 β , Ser9), total GSK3 β , GAPDH, and horseradish-peroxidase- (HRP-) linked anti-rabbit antibody purchased from Cell Signaling Technology (Beverly, MA, USA) were used for Western blot analysis.

Techniques: Inhibition, De-Phosphorylation Assay, Expressing, Activation Assay