Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Drug Design, Development and Therapy

Article Title:

miR-370-3p Alleviates Ulcerative Colitis-Related Colorectal Cancer in Mice Through Inhibiting the Inflammatory Response and Epithelial-Mesenchymal Transition

doi: 10.2147/dddt.s238124

Figure Lengend Snippet: Figure 3 Effect of miR-370-3p on inflammatory response and TLR4 signaling pathway in AOM/DSS-induced mice. (A) The expression of TNF-α, IL-1β, and IL-6 in colonic tissues of mice from the Sham group, UC-CRC group, Ad-control group, and Ad-miR-370-3p group was detected by qRT-PCR. (B) The production of TNF-α and PGE2 in the lysate of the tumor colon tissues were detected by ELISA. (C) The expression of TLR4 and COX-2 in the tumor colon tissues from each group was evaluated by using IHC staining. Scale bar=50 μm. (D) The expression levels of TLR4, COX-2, and pEGFR in the tumor colon tissues from each group were determined by Western blot, and the relative band intensity was analyzed.The comparison among groups was performed by One-way ANOVA. Data are presented as mean ± SD. ##p<0.01 compared with the Sham group, **p<0.01 compared with the Ad-control group.

Article Snippet: After blocking with 5% skim milk (Sangon Biotech, Shanghai China) at room temperature for 1 h, the membranes were incubated with corresponding primary antibodies incuding TLR4 (1:1000, proteintech, Wuhan, China), COX-2 (1:1000, proteintech, Wuhan, China), pEGFR (phosphorylated epidermal growth factor receptor, 1:1000, Affinity, Changzhou, Jiangsu, China), β-catenin (1:5000, proteintech, Wuhan, China), p53 (1:3000, proteintech, Wuhan, China), ki67 (1:1000, Affinity, Changzhou, Jiangsu, China), E-cadherin (1:10,000, proteintech, Wuhan, China), N-cadherin (1:5000, proteintech, Wuhan, China), Vimentin (1:5000, proteintech, Wuhan, China) and GAPDH (1:10,000, proteintech, Wuhan, China) overnight at 4° C. Then the membranes were washed by TBST and incubated with appropriate secondary HRPconjugated goat anti-rabbit or goat anti-mouse antibodies (1:3000, Solarbio, Beijing, China) for 1 h at 37 ° C. The proteins were visualized by using ECL Western Blotting Substrate (Solarbio, Beijing, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot, Comparison

Journal: BMC Neuroscience

Article Title: GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons

doi: 10.1186/1471-2202-11-74

Figure Lengend Snippet: GT1b increases the activity GSK-3β by dephosphorylation in neuron-enriched mesencephalic cultures . (A) Western blot analysis showing dephosphorylation of GSK-3β in GT1b-treated cultures. Cell lysates were analyzed by Western blot analysis with phospho-GSK-3β (Ser9) antibody at indicated time points. After membrane stripping, blots were reprobed with total GSK-3β. (B) The histogram shows quantification of phospho-GSK-3β levels. The values represent the mean ± SEM of four separate experiments; *P < 0.001, significant from non-treated control cultures (0 h) (ANOVA and Student-Newman-Keuls analyses). (C) TH-ip neurons were counted in cultures treated with 20 μg/ml GT1b for 24 h in the absence or presence of 20 μM L803-mt, GSK-3β specific inhibitor. The values represent the mean ± SEM of triplicate cultures in three to five separate samples. *P < 0.001, significant from non-treated control cultures; # P < 0.01, significant from GT1b-treated cultures (ANOVA and Student-Newman-Keuls analyses).

Article Snippet: Materials were purchased from the following companies: mouse anti-tyrosine hydroxylase (TH; Pel-Freez, Rogers, AR, USA), rabbit anti-tyrosine hydroxylase (TH; Pel-Freez), mouse anti-neuron specific nuclear protein (NeuN; Chemicon, Temecula, CA, USA), rabbit anti-GABA and mouse anti-actin (Sigma, St. Louis, MO, USA), rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-phospho-GSK-3β (Ser9), rabbit anti-Akt and rabbit anti-GSK-3β (Cell Signaling Technology, MA, USA), mouse anti-AT8 (phospho-tau (Ser202), INOGENETICS, Gent, Belgium), biotinylated secondary antibody and ABC kit (Vector Laboratory, Burlington, CA, USA), FITC-conjugated anti-mouse IgG and Texas Red-conjugated anti-rabbit IgG (Molecular Probes, OR, USA).

Techniques: Activity Assay, De-Phosphorylation Assay, Western Blot, Membrane, Stripping Membranes, Control

Journal: BMC Neuroscience

Article Title: GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons

doi: 10.1186/1471-2202-11-74

Figure Lengend Snippet: Phosphorylation of tau is increased by GT1b treatment in mesencephalic cultures . Double-immunostaining with AT8 (Ser202; green)) for phospho-tau and TH (red) for DA neurons in mesencephalic cultures treated with vehicle (A) or 20 μg/ml GT1b (B) for 8 h. (D-I) Double-immunostaining showed Tau hyperphosphorylation in the GT1b-injected SN. SN tissues were prepared 72 h after PBS (D-F) or GT1b (60 μg/3 μl, G-I) injection, processed for immunostaining with TH antibody alone (C), double-immunostaining with AT8 (D, G, green) and TH (E, H, red) antibody, and then both images were merged (F, I). Scale bar, 10 μm in A-C, 200 μm in F and 50 μm G-I. SNpc, substantia nigra pars compacta; SNr, substantia nigra reticulata; VTA, ventral tegmental area. (J,K) Cell lysates were analyzed by Western blot analysis with AT8 (Ser202) antibody at indicated time points. After membrane stripping, blots were then reprobed with actin antibody. The histogram shows quantitation of tau phosphorylation. (L.M) Tau phosphorylation is reversed by L803-mt, GSK-3 inhibitor in GT1b-treated mesencephalic cultures. The histogram shows quantitation of tau phosphorylation. The values represent the mean ± SEM of triplicate cultures in three to five separate samples. *P < 0.001, **P < 0.01 significant from non-treated cultures; # P < 0.01, significant from GT1b-treated cultures (ANOVA and Student-Newman-Keuls analyses). C, control; G, GT1b; L, L803mt.

Article Snippet: Materials were purchased from the following companies: mouse anti-tyrosine hydroxylase (TH; Pel-Freez, Rogers, AR, USA), rabbit anti-tyrosine hydroxylase (TH; Pel-Freez), mouse anti-neuron specific nuclear protein (NeuN; Chemicon, Temecula, CA, USA), rabbit anti-GABA and mouse anti-actin (Sigma, St. Louis, MO, USA), rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-phospho-GSK-3β (Ser9), rabbit anti-Akt and rabbit anti-GSK-3β (Cell Signaling Technology, MA, USA), mouse anti-AT8 (phospho-tau (Ser202), INOGENETICS, Gent, Belgium), biotinylated secondary antibody and ABC kit (Vector Laboratory, Burlington, CA, USA), FITC-conjugated anti-mouse IgG and Texas Red-conjugated anti-rabbit IgG (Molecular Probes, OR, USA).

Techniques: Phospho-proteomics, Double Immunostaining, Injection, Immunostaining, Western Blot, Membrane, Stripping Membranes, Quantitation Assay, Control

Journal: BMC Neuroscience

Article Title: GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons

doi: 10.1186/1471-2202-11-74

Figure Lengend Snippet: Schematic drawing of Akt/GSK-3β/tau signaling . Akt is activated in response to ILK or PI3 Kinase signaling, and subsequent activation of Akt. Akt is activated in response to ILK or PI3 kinase signaling, and subsequent activation of Akt. Activated Akt phosphorylates GSK-3β at Ser 9, leading to GSK-3β inactivation. Akt inactivation by GT1b induces dephosphorylation of GSK-3β, leading to GSK-3β activation. GSK-3β activation results in aberrant tau phosphorylation, axon instability, and neuronal death. Although we did not provide direct evidence of whether GT1b neuroxocicity is mediated by ILK or PI3K signaling or both, participation of GT1b in inhibition of Akt upstream signals, such as ILK or PI3K, is predicted.

Article Snippet: Materials were purchased from the following companies: mouse anti-tyrosine hydroxylase (TH; Pel-Freez, Rogers, AR, USA), rabbit anti-tyrosine hydroxylase (TH; Pel-Freez), mouse anti-neuron specific nuclear protein (NeuN; Chemicon, Temecula, CA, USA), rabbit anti-GABA and mouse anti-actin (Sigma, St. Louis, MO, USA), rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-phospho-GSK-3β (Ser9), rabbit anti-Akt and rabbit anti-GSK-3β (Cell Signaling Technology, MA, USA), mouse anti-AT8 (phospho-tau (Ser202), INOGENETICS, Gent, Belgium), biotinylated secondary antibody and ABC kit (Vector Laboratory, Burlington, CA, USA), FITC-conjugated anti-mouse IgG and Texas Red-conjugated anti-rabbit IgG (Molecular Probes, OR, USA).

Techniques: Activation Assay, De-Phosphorylation Assay, Phospho-proteomics, Inhibition

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: Protein levels were assessed using Western blotting following stimulation with IL-6 (10 ng/mL) for durations of 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours in Ca9-22 cells. Densitometric analyses of the Western blot results for ODAM, p-STAT3, STAT3, p-p65, p65, p-IKKα/β, IKKβ, and IKKα are presented. The intensity of the α-tubulin band was normalized to 1, and graphs were generated to illustrate the relative intensities of the other protein bands. IL-6: interleukin-6, ODAM: odontogenic ameloblast-associated protein, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3, p65: nuclear factor-kappa B p65, p-p65: phosphorylated p65, IKKα: IκB kinase α, IKKβ: IκB kinase β, p-IKKα/β: phosphorylated IκB kinase α/β. The data represent the mean ± standard deviation from 3 independent experiments, with * P <0.05 and ** P <0.01 indicating statistical significance.

Article Snippet: These membranes were incubated with primary antibodies: anti-ODAM (PA5-100656; Invitrogen), anti-signal transducer and activator of transcription (STAT) 3 (10253-2-AP; Proteintech, Rosemont, IL, USA), anti-phospho-STAT3 (Tyr705) (ZRB1006; Sigma-Aldrich, St. Louis, MO, USA), anti-nuclear factor-kappa B (NF-κB) p65 (AF5006; Affinity Biosciences, Cincinnati, OH, USA), anti-phosphorylated (phospho-)NF-κB p65 (Ser536) (AF2006; Affinity Biosciences), anti-IκB kinase α (IKKα) (ab32041; Abcam, Cambridge, UK), anti-IκB kinase β (IKKβ) (#2684; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-IKKα/β (Ser176/180) 16A6 (#2697; Cell Signaling Technology), and anti-α-tubulin (sc-5286; Santa Cruz Biotechnology).

Techniques: Western Blot, Generated, Standard Deviation

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: β-catenin is essential for CUG2-induced CSC-like phenotypes. (A) A549-VEC and A549-CUG2 cells were separated into cytosolic and nuclear fractions. β-catenin expression was determined by performing western blot analysis with an anti-β-catenin antibody. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. The cytosolic and nuclear fractions were confirmed by detecting actin and Sp1, respectively. (B) Total RNAs (3 µ g) were isolated from the A549-VECand A549-CUG2 cells, and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (C) A549-VEC and A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector and were harvested at 48 h after the transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results are an average of three experiments; bars indicate the means ± SD ( *** P<0.001, A549-VEC vs A549-CUG2 cells). (D) Transfection of efficiency of β-catenin siRNAs (#1 or #2) was confirmed by western blot analysis. (E) Migration of A549-CUG2 cells was measured with the wound healing assay at 24 h after control or β-catenin siRNAs (#1 and #2) transfection. Wound closure areas were monitored using a phase-contrast microscope at ×100 magnification, and the assay was repeated twice. (F) Invasion of A549-CUG2 cells transfected with control or β-catenin siRNAs (#1 and #2) was determined using the 48-well Boyden chamber. The chamber was assembled using Matrigel-coated polycarbonate filters. Scale bar indicates 100 µ m, and the assay was repeated twice. Each assay was performed in triplicate, and error bars indicate the means ± SD ( *** P<0.001, control vs. β-catenin siRNA). (G) A549-CUG2 cells (1,000 cells per well) transfected with control or β-catenin siRNAs (#1 and #2) were seeded in ultra-low attachment plates for 2, 4, or 6 days. The assay was performed in triplicate, and error bars indicate the means ± SD ( ** P<0.01 and *** P<0.001, control vs. β-catenin siRNA). Scale bars indicate 50 µ m. CUG2, cancer-upregulated gene 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Migration, Wound Healing Assay, Microscopy

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-induced β-catenin phosphorylation is unique and differs from that induced by Wnt3a. (A) A549-VEC and A549-CUG2 cells were treated with wortmannin (10 µ M), H89 (10 µ M), PP2 (2.5 µ M) and DMSO (mock) in the presence of Wnt3a (100 ng/ml) or PBS as a vehicle for 12 h, and cell lysates were prepared. Proteins present in the cell lysates were separated by performing SDS-PAGE on 10% gels, and β-catenin and α-actin were detected using corresponding antibodies. (B and C) Proteins present in the lysates of A549-VEC cells treated with Wnt3a (100 ng/ml) or PBS, and of A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were determined using antibodies against phosphorylated Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (D) Lysates of A549-VEC and A549-CUG2 cells treated with a control or GSK3β siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. GSK3β and phosphorylated GSK3β were detected by performing western blot with corresponding antibodies. CUG2, cancer-upregulated gene 2; GSK3β, glycogen synthase kinase 3β.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Control, Western Blot

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-activated NEK2 is responsible for the phosphorylation of β-catenin at Ser33/Ser37. (A) Lysates of A549-VEC and A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels, and NEK2 was detected by performing western blot analysis with anti-NEK2 antibody. (B) Levels of NEK2 and β-catenin phosphorylated at Ser33/Ser37 (p-β-catenin; p-Ser33/Ser37) in A549-VEC and A549-CUG2 cells were determined by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) β-catenin was pulled down from the lysates of A549-VEC and A549-CUG2 cells by using an anti-β-catenin antibody. NEK2 present in the immunoprecipitates was detected using an anti-NEK2 antibody, and β-catenin present in the immunoprecipitates was detected as a loading control by using an anti-β-catenin antibody. (D) NEK2 was pulled down from A549-VEC and A549-CUG2 cells by using an anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay, including immunoprecipitates as NEK2 kinase, recombinant GST-β-catenin as a substrate, ATP, and a reaction buffer, was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by determining the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing immunoblotting, and β-catenin levels were examined as a loading control. (E) Lysates of A549-CUG2 cells transfected with the control or NEK2 siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (F) Following transfection with control or NEK2 siRNA#1, the levels of p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells were detected by performing immunofluorescence microscopy with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Recombinant, Incubation, Activity Assay, Transfection

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment destabilizes β-catenin in a proteasome- and PKCα- dependent manner. (A) A549-CUG2 cells were treated with CGK062 (0, 1, 10, 50 and 100 µ M) for 24 h, and β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (B) A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector in the presence of CGK062 (50 µ M) and were harvested at 48 h following transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results shown are an average of 3 experiments; bars indicate the means ± SD ( *** P<0.001, 0 vs. 10 µ M, 0 vs. 50 µ M). (C) Total RNAs (3 µ g) were isolated from A549-CUG2 cells treated with or without CGK062 (50 µ M), and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (D) A549-CUG2 cells were treated with MG132 (1 µ M) for 8 h in the absence or presence of CGK062 (50 µ M) and were harvested. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (E) A549-CUG2 cells were treated with BMI (7.5 µ M), a specific PKCα inhibitor, or were transfected with a control and PKCα siRNAs (#1 or #2) in the absence or presence of CGK062 (50 µ M) for 24 h. Transfection efficiency of PKCα siRNAs (#1 or #2) was confirmed by western blot analysis. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. CUG2, cancer-upregulated gene 2; PKC, protein kinase C.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment decreases NEK2 expression and kinase activity in A549-CUG2 cells. (A) Lysates of A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 µ M) for 24 h were separated by performing SDS-PAGE on 10% gels. The expression of NEK2, β-catenin, E-cadherin, N-cadherin and vimentin was detected by performing western blot analysis with corresponding antibodies. (B) Levels of NEK2 and p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO as Mock were detected by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) NEK2 was pulled down from A549-VEC and A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO by using the anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by detecting the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing western blot analysis, and β-catenin levels were examined as a loading control. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Activity Assay, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Incubation, Phospho-proteomics

Journal: PloS one

Article Title: Hydrogen sulfide preconditioning protects rat liver against ischemia/reperfusion injury by activating Akt-GSK-3β signaling and inhibiting mitochondrial permeability transition.

doi: 10.1371/journal.pone.0074422

Figure Lengend Snippet: Figure 8. The effect of preconditioning with 25 μmol/L NaHS on the levels of Bcl-2, p-GSK-3β, and p-Akt. (A) A representative Western blot of Bcl-2. (B) Relative levels of Bcl-2. (C) A representative Western blot of p-GSK-3β and total GSK-3β. (D) Relative levels of p-GSK-3β and total GSK-3β. (E) A representative Western blot of p-Akt and total Akt. (F) Relative levels of p- Akt and total Akt. These experiments were performed in triplicate. The relative densities are expressed as the mean ± SD. * P < 0.05 versus I/R.

Article Snippet: Antibodies for Akt, phosphorylated Akt (p-Akt), GSK3β, phosphorylated GSK-3β (p-GSK-3β), Bcl-2, activated caspase-3/9 and cytochrome c were purchased from Cell Signaling Technology (CST, Boston, MA).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Positive feedback loop of hepatoma-derived growth factor and β-catenin promotes carcinogenesis of colorectal cancer

doi:

Figure Lengend Snippet: A. β-catenin mRNA expression was not significantly altered in HCT116 and HT29 cells with HDGF knockdown by real-time PCR analysis, respectively; B. HDGF knockdown dramatically inhibited β-catenin and c-Myc, cyclin D1 and MMP9 and phos-GSK3β (Ser9) expression in HCT116 cells; C. - D. Nuclear and cytoplasmic β-catenin, c-Myc, cyclin D1, MMP9, and phos-GSK-3β (Ser9) expression were suppressed in HCT116 C. and HT29 D. cells with HDGF knock-down; E. rHDGF enhanced nuclear and cytoplasmic β-catenin, c-Myc, cyclin D1, MMP9, and phos-GSK-3β(Ser9) protein expression in LOVO cells; F. rHDGF reversed the nuclear and cytoplasmic protein expression suppression of β-catenin c-Myc, cyclin D1, MMP9, and phos-GSK-3β (Ser9) in HCT116 upon HDGF knock-down by Western blot analysis. GAPDH was considered as loading control. Lamin B1 was considered as nuclear loading control.

Article Snippet: The membranes were incubated with primary antibodies including rabbit HDGF (Abcam, dilution 1:1000), rabbit β-catenin, rabbit cyclin D1, rabbit c-Myc, rabbit phosphorylated- glycogen synthase kinase 3β (phos-GSK-3β) (Ser9), and rabbit MMP9 (Cell Signaling Technology, Danvers, MA, dilution 1:1000) at 4°C overnight.

Techniques: Expressing, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: TAF-Iβ deficiency inhibits proliferation and promotes apoptosis by rescuing PP2A and inhibiting the AKT/GSK-3β pathway in leukemic cells

doi: 10.3892/etm.2019.8012

Figure Lengend Snippet: TAF-Iβ KD upregulates the expression of PP2A and inhibits the AKT/GSK-3β signaling pathway in NB4 and K562 cells. (A) Expression of PP2A in TAF-Iβ KD and CON groups. (B) Expression of GSK-3β and pGSK-3β(Ser9). (C) Expression of AKT and pAKT(Ser473) in cells with or without SC79 treatment. (D) Expression of GSK-3β and pGSK-3β(Ser9) in cells with or without SC79 treatment. *P<0.05 and **P<0.01 vs. CON. TAF-Iβ, template-activating factor Iβ; AKT, protein kinase B; pAKT(Ser473), Ser473-phosphorylated AKT; KD, knockdown; CON, control; GSK-3β, glycogen synthase kinase-3β; pGSK-3β(Ser9), Ser9-phosphorylated GSK-3β; PP2A, protein phosphatase type 2A.

Article Snippet: The antibodies used were as follows: Anti-TAF-Iβ (cat. no. ab181990, mouse monoclonal, 1:1,000; Abcam, Cambridge, MA, USA), anti-PP2A (cat. no. ab32141, rabbit monoclonal, 1:5,000; Abcam), phosphorylated-glycogen synthase kinase-3β (GSK-3β; Ser9; cat. no. 9336, rabbit monoclonal, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated-AKT (Ser473; cat. no. 9271, rabbit monoclonal, 1:2,000; Cell Signaling Technology, Inc.), anti-caspase-3 (cat. no. 9662), anti-poly(ADP-ribose)polymerase (PARP; cat. no. 9542, rabbit polyclonal, 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. sc-166574, mouse monoclonal, 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Expressing, Knockdown, Control