Journal: eLife

Article Title: CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis

doi: 10.7554/eLife.86689

Figure Lengend Snippet: ( A ) The relative protein levels of CRB3, GSK3-β, and β-catenin in CRB3-depleted MFC10A cells (normalized to β-actin). ( B ) The relative protein levels of CRB3, GSK3-β, and β-catenin in MDA-MB-231 and HCC 1806 breast cancer cells with CRB3b overexpression (normalized to β-actin). The data are three independent experiments performed in triplicate. Bars represent means ± SD; unpaired Student’s t -test, **p<0.01, ***p<0.001.

Article Snippet: The membranes were subjected to immunoblot assays using antibodies against CRB3 (Santa Cruz, sc-292449, both isoforms CRB3a and CRB3b can be detected), GCP2 (Santa Cruz, sc-377117), GCP3 (Santa Cruz, sc-373758), GCP4 (Santa Cruz, sc-271876), GCP5 (Santa Cruz, sc-365837), GCP6 (Abcam, ab95172), γ-tubulin (Proteintech, #66320-1-Ig), Rab11 (Abcam, ab128913), GFP (Roche, #11814460001), Flag (Sigma, F7425), CEP290 (Santa Cruz, sc-390637), GSK3-β (CST, #12456), β-catenin (CST, #8480), GAPDH (Proteintech, #HRP-6004), and β-actin (Proteintech, #HRP-60008).

Techniques: Over Expression

Journal: World Journal of Gastroenterology

Article Title: γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus

doi: 10.3748/wjg.v29.i28.4416

Figure Lengend Snippet: Baclofen inhibits the glycogen synthase kinase 3/β-catenin and signal transducer and activator of transcription 3 pathways. A and B: Phospho-kinase arrays reveal the inhibition of phosphorylation in multiple kinases and signal transducers; C: Glycogen synthase kinase 3 (GSK3)/β-catenin and signal transducer and activator of transcription 3 (STAT3) are included for further analysis as they are key pathways in cholangiocarcinoma (CCA) progression in which β-catenin and STAT3 are common targets of GSK3. RNA expression of γ-aminobutyric acid B2 receptor and that of STAT3 are significantly correlated in clinical CCA samples from Thai patients. HG: High glucose; GABBR2: γ-aminobutyric acid B2 receptor; GSK3: Glycogen synthase kinase 3; STAT3: Signal transducer and activator of transcription 3.

Article Snippet: Primary antibodies used to detect the proteins by western blot were: GABBR2 (1:500, Proteintech, Rosemont, IL), pSTAT3 (Y705) (1:500, Cell Signaling Technology, Danvers, MA), pSTAT3 (S727) (1:500, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), p-glycogen synthase kinase 3 (GSK3)α/β (1:1000, Cell Signaling Technology), GSK3α/β (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz Biotechnology, Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000, Millipore Sigma, Burlington, MA).

Techniques: Inhibition, RNA Expression

Journal: World Journal of Gastroenterology

Article Title: γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus

doi: 10.3748/wjg.v29.i28.4416

Figure Lengend Snippet: Baclofen suppresses the glycogen synthase kinase 3/β-catenin and signal transducer and activators of transcription 3 pathways. A and B: Phosphorylation of glycogen synthase kinase 3 (GSK3) and signal transducer and activators of transcription 3 is decreased after baclofen treatment in both cholangiocarcinoma cell lines, both cultured in normal glucose and high glucose conditions. Total β-catenin protein is also decreased consistently with the decreased phosphorylated GSK3α/β. Western blots show the representative of three biological replications with the same trends of results. Band intensities are the average of three biological replications which are normalized using the intensities of glyceraldehyde-3-phosphate dehydrogenase for each experiment. The levels of phosphorylated forms are normalized with the total forms of their corresponding proteins. NG: Normal glucose; HG: High glucose; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GSK3: Glycogen synthase kinase 3; STAT3: Signal transducer and activator of transcription 3.

Article Snippet: Primary antibodies used to detect the proteins by western blot were: GABBR2 (1:500, Proteintech, Rosemont, IL), pSTAT3 (Y705) (1:500, Cell Signaling Technology, Danvers, MA), pSTAT3 (S727) (1:500, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), p-glycogen synthase kinase 3 (GSK3)α/β (1:1000, Cell Signaling Technology), GSK3α/β (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz Biotechnology, Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000, Millipore Sigma, Burlington, MA).

Techniques: Cell Culture, Western Blot

Journal: World Journal of Gastroenterology

Article Title: γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus

doi: 10.3748/wjg.v29.i28.4416

Figure Lengend Snippet: Schematic summary of the effects of high glucose on γ-aminobutyric acid B2 receptor expression and the effects of baclofen on cholangiocarcinoma cells. High glucose induces the expression of γ-aminobutyric acid B2 receptor (GABBR2) in cholangiocarcinoma (CCA) cells. The treatment of baclofen, a GABBR2 agonist, to CCA cells inhibits phosphorylation of glycogen synthase kinase 3, resulting in the activation of the kinase activity which further phosphorylates β-catenin. Phosphorylated β-catenin is subjected to degradation preventing its function on promoting cell proliferation via c-Myc and cyclin D1 expression. On the other hand, activated GABBR2 by baclofen also inhibits phosphorylation of signal transducer and activator of transcription 3 (STAT3). The inhibition of STAT3 phosphorylation also suppresses its functions as a transcription factor for c-Myc and cyclin D1 expression. Activating GABBR2 by baclofen, thus, suppresses the proliferation of CCA cells. GABBR2: γ-aminobutyric acid B2 receptor; GSK3: Glycogen synthase kinase 3; STAT3: Signal transducer and activator of transcription 3.

Article Snippet: Primary antibodies used to detect the proteins by western blot were: GABBR2 (1:500, Proteintech, Rosemont, IL), pSTAT3 (Y705) (1:500, Cell Signaling Technology, Danvers, MA), pSTAT3 (S727) (1:500, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), p-glycogen synthase kinase 3 (GSK3)α/β (1:1000, Cell Signaling Technology), GSK3α/β (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz Biotechnology, Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000, Millipore Sigma, Burlington, MA).

Techniques: Expressing, Activation Assay, Activity Assay, Inhibition

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: The Journal of Biological Chemistry

Article Title: Carnosic acid inhibits secretion of allergic inflammatory mediators in IgE-activated mast cells via direct regulation of Syk activation

doi: 10.1016/j.jbc.2022.102867

Figure Lengend Snippet: CA treatment inhibits upstream Syk phosphorylation and selectively impairs Akt phosphorylation in allergen-activated BMMCs. Protein isolates from control and CA-treated BMMCs stimulated with TNP-BSA + SCF for 5, 20, and 60 min were analyzed by Western blot. A, representative blots of n = 3 independent primary BMMC cultures showing phospho (p-) Syk, vinculin, p- and total (t-) Akt, GSK3, and AMPK protein expression. Relative protein phosphorylation (phospho/total) was quantified for Syk (Tyr352) (B), Syk (Tyr525/526) (C), Akt (S473) (D), Akt (Thr308) (E), GSK3 (S9) (F), and AMPK (G) in the presence or absence of CA treatment. Data expressed as mean relative phosphorylation ±SEM of three independent primary BMMC cultures. A two-way ANOVA and Dunnett’s multiple comparison were used to determine differences in relative phosphorylation levels following allergen stimulation and CA treatment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, relative to BMMCs not treated with CA (TNP-BSA + SCF only). BMMC, bone marrow–derived mast cell; CA, carnosic acid; SCF, stem cell factor; TNP-BSA, trinitrophenyl-bovine serum albumin.

Article Snippet: Antibodies specific for phospho-Akt (Ser473), phospho-Akt (Thr308) Akt, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2-MAPK, phospho-GSK3β (Ser9), GSK3 (α+β), phospho-IKK α/β (Ser176/180), phospho-IκBα (Ser32/36), phospho-JNK/SAPK (Thr183/Tyr185), JNK-MAPK, Lyn, phospho-p38 (Thr180/Tyr182), p38-MAPK, phospho-Syk (Tyr352), phospho-Syk (Tyr525/526), Syk, phospho-TAK1 (Ser412), phospho-tyrosine (P-Tyr-1000) Multi mAb, and HRP-conjugated anti-rabbit and anti-mouse secondary Abs were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Derivative Assay