signalsilence gsk 3β sirna kit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc signalsilence gsk 3β sirna kit
    <t>GSK-3β</t> inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Signalsilence Gsk 3β Sirna Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/signalsilence gsk 3β sirna kit/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    signalsilence gsk 3β sirna kit - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades"

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-99

    GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Figure Legend Snippet: GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    siRNA targeting of GSK-3β inhibits TNF-α production by LPS-stimulated BV-2 cells . (A) Cells were transfected with GSK-3β-specific (50 and 100 nM) or control siRNA (100 nM) duplexes for 48 h. Endogenous GSK-3β protein was analyzed by western blotting. (B) Parallel cultures were exposed to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control siRNA-transfected cultures. The level of TNF-α in control cultures was 6.08 ± 0.41 ng/ml.
    Figure Legend Snippet: siRNA targeting of GSK-3β inhibits TNF-α production by LPS-stimulated BV-2 cells . (A) Cells were transfected with GSK-3β-specific (50 and 100 nM) or control siRNA (100 nM) duplexes for 48 h. Endogenous GSK-3β protein was analyzed by western blotting. (B) Parallel cultures were exposed to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control siRNA-transfected cultures. The level of TNF-α in control cultures was 6.08 ± 0.41 ng/ml.

    Techniques Used: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    Blockade of GSK-3β decreases NF-κB transcriptional activity . BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods . (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with LPS alone.
    Figure Legend Snippet: Blockade of GSK-3β decreases NF-κB transcriptional activity . BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods . (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with LPS alone.

    Techniques Used: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Luciferase, Construct

    GSK-3β inactivation inhibits NF-κB acetylation at Lys310 but not phosphorylation . BV-2 cells (A) or primary microglia (B and C) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were prepared and subjected to western blotting using antibodies specific for phosphorylated (Ser276, 468 and 536), acetylated (Lys310) or total forms of p65. The immunoblots are representative of three independent experiments. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.
    Figure Legend Snippet: GSK-3β inactivation inhibits NF-κB acetylation at Lys310 but not phosphorylation . BV-2 cells (A) or primary microglia (B and C) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were prepared and subjected to western blotting using antibodies specific for phosphorylated (Ser276, 468 and 536), acetylated (Lys310) or total forms of p65. The immunoblots are representative of three independent experiments. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Techniques Used: Western Blot

    Inhibition of GSK-3β activity blocks LPS-induced JNK signaling . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods . Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with LPS alone.
    Figure Legend Snippet: Inhibition of GSK-3β activity blocks LPS-induced JNK signaling . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods . Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with LPS alone.

    Techniques Used: Inhibition, Activity Assay, Isolation, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Luciferase, Construct

    SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells . Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.
    Figure Legend Snippet: SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells . Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.

    Techniques Used: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase

    Decreased GSK-3β activity blocks MLK3 signaling . BV-2 cells (A) or primary microglia (B) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times (A) or 30 min (B). Western analysis was used to determine total and phosphorylated c-Jun, JNK, MKK4 and MLK3 proteins in whole cell extracts. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.
    Figure Legend Snippet: Decreased GSK-3β activity blocks MLK3 signaling . BV-2 cells (A) or primary microglia (B) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times (A) or 30 min (B). Western analysis was used to determine total and phosphorylated c-Jun, JNK, MKK4 and MLK3 proteins in whole cell extracts. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Techniques Used: Activity Assay, Western Blot

    Association of MLK3 and GSK-3β . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.
    Figure Legend Snippet: Association of MLK3 and GSK-3β . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot

    GSK-3β-mediated NF-κB and MLK3/JNK are two independent signaling pathways in the induction of TNF-α . (A) BV-2 cells were pretreated with SP600125 (SP, 10 μM), K252a (50 nM), BAY 11-7082 (BAY, 5 μM) or PDTC (20 μM) for 30 min following exposure to 100 ng/ml LPS for another 30 min. IκB-α degradation was determined by western blotting. (B) NF-κB luciferase reporter assays were performed by LPS stimulation in the absence or presence of 10 μM SP600125, 50 nM K252a or 20 μM PDTC (6 h) of BV-2 cells as described in Fig. 3. Data are presented as mean + SEM for three independent experiments. ** p < 0.01 compared with LPS alone. (C) Cells were treated as described in (A). Phosphorylated and total JNK and c-Jun were detected by western blotting in whole cell extracts. (D) Cells were preincubated with SP600125 (2.5 μM), K252a (12.5 nM), BAY 11-7082 (0.625 μM), SP600125 plus BAY 11-7082 or K252a plus BAY 11-7082 for 30 min, and then treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. # p , ## p or * p < 0.01 compared with SP600125, K252a or BAY 11-7082 treated cells, respectively. The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 5.74 ± 0.41 ng/ml.
    Figure Legend Snippet: GSK-3β-mediated NF-κB and MLK3/JNK are two independent signaling pathways in the induction of TNF-α . (A) BV-2 cells were pretreated with SP600125 (SP, 10 μM), K252a (50 nM), BAY 11-7082 (BAY, 5 μM) or PDTC (20 μM) for 30 min following exposure to 100 ng/ml LPS for another 30 min. IκB-α degradation was determined by western blotting. (B) NF-κB luciferase reporter assays were performed by LPS stimulation in the absence or presence of 10 μM SP600125, 50 nM K252a or 20 μM PDTC (6 h) of BV-2 cells as described in Fig. 3. Data are presented as mean + SEM for three independent experiments. ** p < 0.01 compared with LPS alone. (C) Cells were treated as described in (A). Phosphorylated and total JNK and c-Jun were detected by western blotting in whole cell extracts. (D) Cells were preincubated with SP600125 (2.5 μM), K252a (12.5 nM), BAY 11-7082 (0.625 μM), SP600125 plus BAY 11-7082 or K252a plus BAY 11-7082 for 30 min, and then treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. # p , ## p or * p < 0.01 compared with SP600125, K252a or BAY 11-7082 treated cells, respectively. The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 5.74 ± 0.41 ng/ml.

    Techniques Used: Western Blot, Luciferase, Enzyme-linked Immunosorbent Assay

    signalsilence gsk 3β sirna kit  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 94

    Structured Review

    Cell Signaling Technology Inc signalsilence gsk 3β sirna kit
    <t>GSK-3β</t> inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Signalsilence Gsk 3β Sirna Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/signalsilence gsk 3β sirna kit/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    signalsilence gsk 3β sirna kit - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades"

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-99

    GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Figure Legend Snippet: GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    siRNA targeting of GSK-3β inhibits TNF-α production by LPS-stimulated BV-2 cells . (A) Cells were transfected with GSK-3β-specific (50 and 100 nM) or control siRNA (100 nM) duplexes for 48 h. Endogenous GSK-3β protein was analyzed by western blotting. (B) Parallel cultures were exposed to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control siRNA-transfected cultures. The level of TNF-α in control cultures was 6.08 ± 0.41 ng/ml.
    Figure Legend Snippet: siRNA targeting of GSK-3β inhibits TNF-α production by LPS-stimulated BV-2 cells . (A) Cells were transfected with GSK-3β-specific (50 and 100 nM) or control siRNA (100 nM) duplexes for 48 h. Endogenous GSK-3β protein was analyzed by western blotting. (B) Parallel cultures were exposed to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control siRNA-transfected cultures. The level of TNF-α in control cultures was 6.08 ± 0.41 ng/ml.

    Techniques Used: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    Blockade of GSK-3β decreases NF-κB transcriptional activity . BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods . (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with LPS alone.
    Figure Legend Snippet: Blockade of GSK-3β decreases NF-κB transcriptional activity . BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods . (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with LPS alone.

    Techniques Used: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Luciferase, Construct

    GSK-3β inactivation inhibits NF-κB acetylation at Lys310 but not phosphorylation . BV-2 cells (A) or primary microglia (B and C) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were prepared and subjected to western blotting using antibodies specific for phosphorylated (Ser276, 468 and 536), acetylated (Lys310) or total forms of p65. The immunoblots are representative of three independent experiments. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.
    Figure Legend Snippet: GSK-3β inactivation inhibits NF-κB acetylation at Lys310 but not phosphorylation . BV-2 cells (A) or primary microglia (B and C) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were prepared and subjected to western blotting using antibodies specific for phosphorylated (Ser276, 468 and 536), acetylated (Lys310) or total forms of p65. The immunoblots are representative of three independent experiments. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Techniques Used: Western Blot

    Inhibition of GSK-3β activity blocks LPS-induced JNK signaling . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods . Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with LPS alone.
    Figure Legend Snippet: Inhibition of GSK-3β activity blocks LPS-induced JNK signaling . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods . Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with LPS alone.

    Techniques Used: Inhibition, Activity Assay, Isolation, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Luciferase, Construct

    SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells . Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.
    Figure Legend Snippet: SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells . Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.

    Techniques Used: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase

    Decreased GSK-3β activity blocks MLK3 signaling . BV-2 cells (A) or primary microglia (B) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times (A) or 30 min (B). Western analysis was used to determine total and phosphorylated c-Jun, JNK, MKK4 and MLK3 proteins in whole cell extracts. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.
    Figure Legend Snippet: Decreased GSK-3β activity blocks MLK3 signaling . BV-2 cells (A) or primary microglia (B) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times (A) or 30 min (B). Western analysis was used to determine total and phosphorylated c-Jun, JNK, MKK4 and MLK3 proteins in whole cell extracts. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Techniques Used: Activity Assay, Western Blot

    Association of MLK3 and GSK-3β . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.
    Figure Legend Snippet: Association of MLK3 and GSK-3β . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot

    GSK-3β-mediated NF-κB and MLK3/JNK are two independent signaling pathways in the induction of TNF-α . (A) BV-2 cells were pretreated with SP600125 (SP, 10 μM), K252a (50 nM), BAY 11-7082 (BAY, 5 μM) or PDTC (20 μM) for 30 min following exposure to 100 ng/ml LPS for another 30 min. IκB-α degradation was determined by western blotting. (B) NF-κB luciferase reporter assays were performed by LPS stimulation in the absence or presence of 10 μM SP600125, 50 nM K252a or 20 μM PDTC (6 h) of BV-2 cells as described in Fig. 3. Data are presented as mean + SEM for three independent experiments. ** p < 0.01 compared with LPS alone. (C) Cells were treated as described in (A). Phosphorylated and total JNK and c-Jun were detected by western blotting in whole cell extracts. (D) Cells were preincubated with SP600125 (2.5 μM), K252a (12.5 nM), BAY 11-7082 (0.625 μM), SP600125 plus BAY 11-7082 or K252a plus BAY 11-7082 for 30 min, and then treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. # p , ## p or * p < 0.01 compared with SP600125, K252a or BAY 11-7082 treated cells, respectively. The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 5.74 ± 0.41 ng/ml.
    Figure Legend Snippet: GSK-3β-mediated NF-κB and MLK3/JNK are two independent signaling pathways in the induction of TNF-α . (A) BV-2 cells were pretreated with SP600125 (SP, 10 μM), K252a (50 nM), BAY 11-7082 (BAY, 5 μM) or PDTC (20 μM) for 30 min following exposure to 100 ng/ml LPS for another 30 min. IκB-α degradation was determined by western blotting. (B) NF-κB luciferase reporter assays were performed by LPS stimulation in the absence or presence of 10 μM SP600125, 50 nM K252a or 20 μM PDTC (6 h) of BV-2 cells as described in Fig. 3. Data are presented as mean + SEM for three independent experiments. ** p < 0.01 compared with LPS alone. (C) Cells were treated as described in (A). Phosphorylated and total JNK and c-Jun were detected by western blotting in whole cell extracts. (D) Cells were preincubated with SP600125 (2.5 μM), K252a (12.5 nM), BAY 11-7082 (0.625 μM), SP600125 plus BAY 11-7082 or K252a plus BAY 11-7082 for 30 min, and then treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. # p , ## p or * p < 0.01 compared with SP600125, K252a or BAY 11-7082 treated cells, respectively. The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 5.74 ± 0.41 ng/ml.

    Techniques Used: Western Blot, Luciferase, Enzyme-linked Immunosorbent Assay

    gsk 3α β sirna signalsilence kit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gsk 3α β
    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot <t>of</t> <t>total</t> <t>GSK-3α/β,</t> phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
    Gsk 3α β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling"

    Article Title: TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-018-1310-7

    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
    Figure Legend Snippet: (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).

    Techniques Used: Western Blot, Expressing, Activation Assay, Blocking Assay

    p gsk 3α β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p gsk 3α β
    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot <t>of</t> <t>total</t> <t>GSK-3α/β,</t> phosphorylated GSK-3α/β <t>(Ser21/9),</t> and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
    P Gsk 3α β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling"

    Article Title: TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-018-1310-7

    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
    Figure Legend Snippet: (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).

    Techniques Used: Western Blot, Expressing, Activation Assay, Blocking Assay

    signalsilence gsk3a b sirna kit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc signalsilence gsk3a b sirna kit
    Signalsilence Gsk3a B Sirna Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti gsk 3α sirna  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti gsk 3α sirna
    <t>siRNA-Mediated</t> Silencing of <t>GSK-3α</t> and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells
    Anti Gsk 3α Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells"

    Article Title: 6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2016.10.017

    siRNA-Mediated Silencing of GSK-3α and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells
    Figure Legend Snippet: siRNA-Mediated Silencing of GSK-3α and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells

    Techniques Used: Expressing

    signalsilence gsk 3α β sirna  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc signalsilence gsk 3α β sirna
    Signalsilence Gsk 3α β Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sirna targeting gsk3 α β isoforms  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sirna targeting gsk3 α β isoforms
    <t>GSK3</t> signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3 . Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.
    Sirna Targeting Gsk3 α β Isoforms, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics"

    Article Title: GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201406020

    GSK3 signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3 . Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.
    Figure Legend Snippet: GSK3 signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3 . Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.

    Techniques Used: Staining, Immunofluorescence, Transfection, Fluorescence, Western Blot, Stable Transfection, Expressing

    GSK3 is recruited to DP to modulate DP–IF complexes. (A) Endogenous DP associates with HA-GSK3 in HA immunoprecipitations in HEK 293 cells, assessed by immunoblotting. (B) SCC9 cells were fixed, and DP and GSK3 associations were analyzed by PLA analysis using anti-DP (NW6) and anti-GSK3 antibodies. Direct interactions (red) and nuclei (DAPI, blue). Bars, 10 µm. (B′) PLA fluorescent spots are counted and divided by the total number of nuclei in an image. Data were obtained from antibody pairs for >500 cells per experiment for three independent experiments. *, P < 0.001. Error bars indicate SEM. (C and C′) Phospho-DP in LiCl- or NaCl-treated SCC9s, assessed by immunoblotting. Full-length endogenous DP has two known isoforms, DPI (310 kD) and DPII (250 kD) that are generated by alternative splicing. Both of these contain S2849 and are recognized by the phospho-DP antibody. Densitometry quantification of three independent experiments. Error bars indicate SEM. (D) DP phosphorylation in siCtr or siGSK3-treated cells, assessed by immunoblotting. (E and E′) Phospho-DP in control or caGSK3 transfected cells, and lysates were assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (F) S-tag affinity pull-down of HA-GSK3 and DP S-tag (DP CT) HEK 293 cells, assessed by immunoblotting. (G) NaCl-, LiCl-, or Ly-294,002 (GSK3 activator)–treated DP S-tag (DP CT) expressing cell lysates, assessed by immunoblotting. DP S-tag (DP CT) is present as a doublet where the lower band (32 kD) contains phosphorylation of S2849, whereas the higher band (red arrow, 36 kD) contains both phosphorylation of S2849 and downstream GSK3-phosphorylated sites (see cartoon in ). (G′) Densitometry quantification of G from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) NaCl- or LiCl-treated DP S-tag (DP CT) cells were transfected with control or caGSK3, assessed by immunoblotting. Long exposure and short exposure of DP CT blot shown to visualize upper phospho-states of DP (36 kD). (H′) Densitometry quantification of phospho-DP divided by total DP from three independent experiments. Error bars indicate SEM.
    Figure Legend Snippet: GSK3 is recruited to DP to modulate DP–IF complexes. (A) Endogenous DP associates with HA-GSK3 in HA immunoprecipitations in HEK 293 cells, assessed by immunoblotting. (B) SCC9 cells were fixed, and DP and GSK3 associations were analyzed by PLA analysis using anti-DP (NW6) and anti-GSK3 antibodies. Direct interactions (red) and nuclei (DAPI, blue). Bars, 10 µm. (B′) PLA fluorescent spots are counted and divided by the total number of nuclei in an image. Data were obtained from antibody pairs for >500 cells per experiment for three independent experiments. *, P < 0.001. Error bars indicate SEM. (C and C′) Phospho-DP in LiCl- or NaCl-treated SCC9s, assessed by immunoblotting. Full-length endogenous DP has two known isoforms, DPI (310 kD) and DPII (250 kD) that are generated by alternative splicing. Both of these contain S2849 and are recognized by the phospho-DP antibody. Densitometry quantification of three independent experiments. Error bars indicate SEM. (D) DP phosphorylation in siCtr or siGSK3-treated cells, assessed by immunoblotting. (E and E′) Phospho-DP in control or caGSK3 transfected cells, and lysates were assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (F) S-tag affinity pull-down of HA-GSK3 and DP S-tag (DP CT) HEK 293 cells, assessed by immunoblotting. (G) NaCl-, LiCl-, or Ly-294,002 (GSK3 activator)–treated DP S-tag (DP CT) expressing cell lysates, assessed by immunoblotting. DP S-tag (DP CT) is present as a doublet where the lower band (32 kD) contains phosphorylation of S2849, whereas the higher band (red arrow, 36 kD) contains both phosphorylation of S2849 and downstream GSK3-phosphorylated sites (see cartoon in ). (G′) Densitometry quantification of G from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) NaCl- or LiCl-treated DP S-tag (DP CT) cells were transfected with control or caGSK3, assessed by immunoblotting. Long exposure and short exposure of DP CT blot shown to visualize upper phospho-states of DP (36 kD). (H′) Densitometry quantification of phospho-DP divided by total DP from three independent experiments. Error bars indicate SEM.

    Techniques Used: Western Blot, Generated, Transfection, Expressing

    GSK3 promotes phosphorylation cascades of the DP CT tail. (A) GSK3 cascade 1 (solid arrows) and 2 (dotted arrows) in WT, S2849G (red), and R2834H (*) DP. (B) NaCl- or LiCl-treated WT DP S-tag (DP CT) cell lysates were assessed by phos-tag immunoblot analyses (red arrow, 36 kD). (C and C′) Phosphorylation of DP S-tag (DP CT) point mutations, S2845A and T2853A, assessed by immunoblotting. Red arrows mark GSK3 phosphorylation of DP. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (D and D′) NaCl-, LiCl-, DMSO-, or Ly-294,002–treated WT, S2845A, or T2853A DP S-tag (DP CT) lysates, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (E and E′) DMSO-, KT 5720-, Gö 6976-, or LiCl-treated DP S-tag (DP CT) lysates, assessed by immunoblotting (red arrow, 36 kD). Densitometry quantification of three independent experiments. Error bars indicate SEM. (F) DP S-tag (DP CT) expressing HEK 293 cells were treated with kinase inhibitors targeting CKI, CKII, MNKI, atypical PKC, and DYRK kinases, assessed by immunoblotting. (G) Control, 40 mM LiCl-, and PMA-treated SCC9s, assessed by immunofluorescence. Bars, 10 µm. (H and H′) GSK3 interactions with S2849G and WT DP S-tag (DP CT) in HEK 293 cells were examined after S-tag pull-down analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting and quantified by densitometry. *, P < 0.03. Error bars indicate SEM.
    Figure Legend Snippet: GSK3 promotes phosphorylation cascades of the DP CT tail. (A) GSK3 cascade 1 (solid arrows) and 2 (dotted arrows) in WT, S2849G (red), and R2834H (*) DP. (B) NaCl- or LiCl-treated WT DP S-tag (DP CT) cell lysates were assessed by phos-tag immunoblot analyses (red arrow, 36 kD). (C and C′) Phosphorylation of DP S-tag (DP CT) point mutations, S2845A and T2853A, assessed by immunoblotting. Red arrows mark GSK3 phosphorylation of DP. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (D and D′) NaCl-, LiCl-, DMSO-, or Ly-294,002–treated WT, S2845A, or T2853A DP S-tag (DP CT) lysates, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (E and E′) DMSO-, KT 5720-, Gö 6976-, or LiCl-treated DP S-tag (DP CT) lysates, assessed by immunoblotting (red arrow, 36 kD). Densitometry quantification of three independent experiments. Error bars indicate SEM. (F) DP S-tag (DP CT) expressing HEK 293 cells were treated with kinase inhibitors targeting CKI, CKII, MNKI, atypical PKC, and DYRK kinases, assessed by immunoblotting. (G) Control, 40 mM LiCl-, and PMA-treated SCC9s, assessed by immunofluorescence. Bars, 10 µm. (H and H′) GSK3 interactions with S2849G and WT DP S-tag (DP CT) in HEK 293 cells were examined after S-tag pull-down analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting and quantified by densitometry. *, P < 0.03. Error bars indicate SEM.

    Techniques Used: Western Blot, Expressing, Immunofluorescence

    AC mutation alters DP phosphorylation by GSK3. (A) GSK3 interactions with R2834H and WT DP S-tag (DP CT) in HEK 293 cells were examined following S-tag pulldown analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting. (A′) Densitometry quantification of GSK3 interactions with WT and R2834H DP S-tag (DP CT). *, P < 0.03. Error bars indicate SEM. (B) PLA of WT, R2834H, or S2849G DP-GFP cells incubated with primary antibody pairs of GFP-GSK3. Nuclei were stained with DAPI (blue). (B′) PLA fluorescence intensity divided by number of nuclei was performed for each cell line using primary antibody pairs of GFP-GSK3 and with controls GSK3-RIgG or GFP-MIgG. Data quantification was analyzed from three separate experiments (>500 nuclei per experiment). *, P < 0.05. Error bars indicate SEM. (C) Control or caGSK3-transfected and LiCl- or Ly-294,002 (GSK3 Act.)–treated DP-GFP cell lines, assessed by immunofluorescence. Bars, 10 µm. (D and D′) R2834H DP S-tag (DP CT) cells were treated with NaCl or LiCl or transfected with caGSK3, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM.
    Figure Legend Snippet: AC mutation alters DP phosphorylation by GSK3. (A) GSK3 interactions with R2834H and WT DP S-tag (DP CT) in HEK 293 cells were examined following S-tag pulldown analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting. (A′) Densitometry quantification of GSK3 interactions with WT and R2834H DP S-tag (DP CT). *, P < 0.03. Error bars indicate SEM. (B) PLA of WT, R2834H, or S2849G DP-GFP cells incubated with primary antibody pairs of GFP-GSK3. Nuclei were stained with DAPI (blue). (B′) PLA fluorescence intensity divided by number of nuclei was performed for each cell line using primary antibody pairs of GFP-GSK3 and with controls GSK3-RIgG or GFP-MIgG. Data quantification was analyzed from three separate experiments (>500 nuclei per experiment). *, P < 0.05. Error bars indicate SEM. (C) Control or caGSK3-transfected and LiCl- or Ly-294,002 (GSK3 Act.)–treated DP-GFP cell lines, assessed by immunofluorescence. Bars, 10 µm. (D and D′) R2834H DP S-tag (DP CT) cells were treated with NaCl or LiCl or transfected with caGSK3, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM.

    Techniques Used: Mutagenesis, Western Blot, Incubation, Staining, Fluorescence, Transfection, Immunofluorescence

    GSK3 phosphorylation of DP altered in R2834H DP mice. (A) Transgenic mouse hearts were lysed in urea sample buffer and phosphorylation of WT and R2834H DP-Flag were analyzed by immunoblotting using DP phospho-specific and GSK3 substrate phospho-specific antibodies. Total levels of DP expression were assessed using anti-DP (NW6) and Flag. Tubulin was used as a loading control. (A′) Densitometry quantification indicates that although total levels of DP expression are similar, phosphorylation of R2834H DP was dramatically decreased compared with WT DP using both the DP-specific phospho-antibody and a GSK3 substrate-specific phospho-antibody. The data shown are from a single representative experiment out of three repeats ( n = 3). Error bars represent SEM. *, P < 0.001. (B) R2834H DP-trangenic mice demonstrate altered cytoplasmic localization of DP and disruption of cell–cell contacts in cardiac tissues, assessed by confocal immunofluorescence. WT and R2834H DP cardiac sections were stained with anti-Flag and anti-desmin antibodies. (C) PLA analyses were performed with WT and R2834H DP cardiac sections incubated with primary antibody pairs DP (NW6)-GSK3, DP (NW6)-MIgG, or GSK3-RIgG. Colocalization allows for hybridization, ligation, and amplification of oligonucleotide adducts fused to secondary antibodies, ultimately producing a fluorescent spot (red) in situ. Blue DAPI staining marks nuclei. Bars, 10 µm. (C′) For the quantification of PLA, PLA spots were counted and divided by total number of nuclei in a frame. Quantification shows a statistically significant enhancement of signal for the DP-GSK3 antibody pairing in WT hearts compared with R2834H transgenic mouse hearts. *, P < 0.03; **, P < 0.001; from >500 cells counted from four independent experiments. Error bars indicate SEM.
    Figure Legend Snippet: GSK3 phosphorylation of DP altered in R2834H DP mice. (A) Transgenic mouse hearts were lysed in urea sample buffer and phosphorylation of WT and R2834H DP-Flag were analyzed by immunoblotting using DP phospho-specific and GSK3 substrate phospho-specific antibodies. Total levels of DP expression were assessed using anti-DP (NW6) and Flag. Tubulin was used as a loading control. (A′) Densitometry quantification indicates that although total levels of DP expression are similar, phosphorylation of R2834H DP was dramatically decreased compared with WT DP using both the DP-specific phospho-antibody and a GSK3 substrate-specific phospho-antibody. The data shown are from a single representative experiment out of three repeats ( n = 3). Error bars represent SEM. *, P < 0.001. (B) R2834H DP-trangenic mice demonstrate altered cytoplasmic localization of DP and disruption of cell–cell contacts in cardiac tissues, assessed by confocal immunofluorescence. WT and R2834H DP cardiac sections were stained with anti-Flag and anti-desmin antibodies. (C) PLA analyses were performed with WT and R2834H DP cardiac sections incubated with primary antibody pairs DP (NW6)-GSK3, DP (NW6)-MIgG, or GSK3-RIgG. Colocalization allows for hybridization, ligation, and amplification of oligonucleotide adducts fused to secondary antibodies, ultimately producing a fluorescent spot (red) in situ. Blue DAPI staining marks nuclei. Bars, 10 µm. (C′) For the quantification of PLA, PLA spots were counted and divided by total number of nuclei in a frame. Quantification shows a statistically significant enhancement of signal for the DP-GSK3 antibody pairing in WT hearts compared with R2834H transgenic mouse hearts. *, P < 0.03; **, P < 0.001; from >500 cells counted from four independent experiments. Error bars indicate SEM.

    Techniques Used: Transgenic Assay, Western Blot, Expressing, Immunofluorescence, Staining, Incubation, Hybridization, Ligation, Amplification, In Situ

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    Cell Signaling Technology Inc signalsilence gsk 3β sirna kit
    <t>GSK-3β</t> inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Signalsilence Gsk 3β Sirna Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk 3α β sirna signalsilence kit
    <t>GSK-3β</t> inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Gsk 3α β Sirna Signalsilence Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk 3α β sirna
    <t>GSK-3β</t> inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
    Gsk 3α β Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc signalsilences gsk 3a b sirna
    <t>GSK-3β</t> inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.
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    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot <t>of</t> <t>total</t> <t>GSK-3α/β,</t> phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
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    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot <t>of</t> <t>total</t> <t>GSK-3α/β,</t> phosphorylated GSK-3α/β <t>(Ser21/9),</t> and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
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    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot <t>of</t> <t>total</t> <t>GSK-3α/β,</t> phosphorylated GSK-3α/β <t>(Ser21/9),</t> and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).
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    <t>siRNA-Mediated</t> Silencing of <t>GSK-3α</t> and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells
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    <t>siRNA-Mediated</t> Silencing of <t>GSK-3α</t> and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells
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    <t>GSK3</t> signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3 . Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.
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    Image Search Results


    GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia . (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods . The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    siRNA targeting of GSK-3β inhibits TNF-α production by LPS-stimulated BV-2 cells . (A) Cells were transfected with GSK-3β-specific (50 and 100 nM) or control siRNA (100 nM) duplexes for 48 h. Endogenous GSK-3β protein was analyzed by western blotting. (B) Parallel cultures were exposed to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control siRNA-transfected cultures. The level of TNF-α in control cultures was 6.08 ± 0.41 ng/ml.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: siRNA targeting of GSK-3β inhibits TNF-α production by LPS-stimulated BV-2 cells . (A) Cells were transfected with GSK-3β-specific (50 and 100 nM) or control siRNA (100 nM) duplexes for 48 h. Endogenous GSK-3β protein was analyzed by western blotting. (B) Parallel cultures were exposed to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with control siRNA-transfected cultures. The level of TNF-α in control cultures was 6.08 ± 0.41 ng/ml.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    Blockade of GSK-3β decreases NF-κB transcriptional activity . BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods . (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with LPS alone.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: Blockade of GSK-3β decreases NF-κB transcriptional activity . BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods . (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with LPS alone.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Luciferase, Construct

    GSK-3β inactivation inhibits NF-κB acetylation at Lys310 but not phosphorylation . BV-2 cells (A) or primary microglia (B and C) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were prepared and subjected to western blotting using antibodies specific for phosphorylated (Ser276, 468 and 536), acetylated (Lys310) or total forms of p65. The immunoblots are representative of three independent experiments. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: GSK-3β inactivation inhibits NF-κB acetylation at Lys310 but not phosphorylation . BV-2 cells (A) or primary microglia (B and C) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were prepared and subjected to western blotting using antibodies specific for phosphorylated (Ser276, 468 and 536), acetylated (Lys310) or total forms of p65. The immunoblots are representative of three independent experiments. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Western Blot

    Inhibition of GSK-3β activity blocks LPS-induced JNK signaling . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods . Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with LPS alone.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: Inhibition of GSK-3β activity blocks LPS-induced JNK signaling . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods . Data are presented as mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with LPS alone.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Inhibition, Activity Assay, Isolation, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Luciferase, Construct

    SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells . Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells . Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase

    Decreased GSK-3β activity blocks MLK3 signaling . BV-2 cells (A) or primary microglia (B) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times (A) or 30 min (B). Western analysis was used to determine total and phosphorylated c-Jun, JNK, MKK4 and MLK3 proteins in whole cell extracts. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: Decreased GSK-3β activity blocks MLK3 signaling . BV-2 cells (A) or primary microglia (B) were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times (A) or 30 min (B). Western analysis was used to determine total and phosphorylated c-Jun, JNK, MKK4 and MLK3 proteins in whole cell extracts. Data are presented as mean ± SEM for three independent experiments. ** p < 0.01 compared with respective cultures treated with LPS alone.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Activity Assay, Western Blot

    Association of MLK3 and GSK-3β . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: Association of MLK3 and GSK-3β . BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Immunoprecipitation, SDS Page, Western Blot

    GSK-3β-mediated NF-κB and MLK3/JNK are two independent signaling pathways in the induction of TNF-α . (A) BV-2 cells were pretreated with SP600125 (SP, 10 μM), K252a (50 nM), BAY 11-7082 (BAY, 5 μM) or PDTC (20 μM) for 30 min following exposure to 100 ng/ml LPS for another 30 min. IκB-α degradation was determined by western blotting. (B) NF-κB luciferase reporter assays were performed by LPS stimulation in the absence or presence of 10 μM SP600125, 50 nM K252a or 20 μM PDTC (6 h) of BV-2 cells as described in Fig. 3. Data are presented as mean + SEM for three independent experiments. ** p < 0.01 compared with LPS alone. (C) Cells were treated as described in (A). Phosphorylated and total JNK and c-Jun were detected by western blotting in whole cell extracts. (D) Cells were preincubated with SP600125 (2.5 μM), K252a (12.5 nM), BAY 11-7082 (0.625 μM), SP600125 plus BAY 11-7082 or K252a plus BAY 11-7082 for 30 min, and then treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. # p , ## p or * p < 0.01 compared with SP600125, K252a or BAY 11-7082 treated cells, respectively. The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 5.74 ± 0.41 ng/ml.

    Journal: Journal of Neuroinflammation

    Article Title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    doi: 10.1186/1742-2094-7-99

    Figure Lengend Snippet: GSK-3β-mediated NF-κB and MLK3/JNK are two independent signaling pathways in the induction of TNF-α . (A) BV-2 cells were pretreated with SP600125 (SP, 10 μM), K252a (50 nM), BAY 11-7082 (BAY, 5 μM) or PDTC (20 μM) for 30 min following exposure to 100 ng/ml LPS for another 30 min. IκB-α degradation was determined by western blotting. (B) NF-κB luciferase reporter assays were performed by LPS stimulation in the absence or presence of 10 μM SP600125, 50 nM K252a or 20 μM PDTC (6 h) of BV-2 cells as described in Fig. 3. Data are presented as mean + SEM for three independent experiments. ** p < 0.01 compared with LPS alone. (C) Cells were treated as described in (A). Phosphorylated and total JNK and c-Jun were detected by western blotting in whole cell extracts. (D) Cells were preincubated with SP600125 (2.5 μM), K252a (12.5 nM), BAY 11-7082 (0.625 μM), SP600125 plus BAY 11-7082 or K252a plus BAY 11-7082 for 30 min, and then treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. # p , ## p or * p < 0.01 compared with SP600125, K252a or BAY 11-7082 treated cells, respectively. The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 5.74 ± 0.41 ng/ml.

    Article Snippet: Murine GSK-3β was targeted with a small interfering RNA (siRNA) duplex supplied by SignalSilence ® GSK-3β siRNA kit (Cell Signaling), according to the protocol of the manufacturer.

    Techniques: Western Blot, Luciferase, Enzyme-linked Immunosorbent Assay

    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).

    Journal: Molecular neurobiology

    Article Title: TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling

    doi: 10.1007/s12035-018-1310-7

    Figure Lengend Snippet: (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).

    Article Snippet: Primary antisera used were non-phosphorylated “active” β-catenin (Ser33/37/Thr41, Cell Signaling), GSK-3α/β (Cell Signaling), p-GSK-3α/β (Ser21/9, Cell Signaling), pAkt (Ser473, Cell Signaling), pan-Akt (Cell Signaling), and β-actin (Cell Signaling).

    Techniques: Western Blot, Expressing, Activation Assay, Blocking Assay

    (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).

    Journal: Molecular neurobiology

    Article Title: TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling

    doi: 10.1007/s12035-018-1310-7

    Figure Lengend Snippet: (A) Western blot of phosphorylated (Ser473) Akt and total Akt expression in primary OPC cultures vehicle treated (PBS), treated with rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for 2 hours in differentiation media. Relative changes in Akt activation (pAkt) in each treatment conditioned was quantified relative to Akt levels (lower panel) from the same blot. Each lane represents an independent measurement. (B) Quantification of Akt activation in OPCs in response to treatments (one-way ANOVA, P < 0.0001; *, P < 0.05 PBS vs rm-TIMP-1; **, P < 0.01, PBS vs MK2006 or MK2206 + rm-TIMP-1). (C) Western blot of total GSK-3α/β, phosphorylated GSK-3α/β (Ser21/9), and GAPDH in OPC cultures treated with PBS, rm-TIMP-1 (10 ng/mL), Akt inhibitor: MK2206 alone (5 μΜ), or rm-TIMP-1 with MK2206 for one hour in differentiation media. α at 51 kDa and β at 47 kDa. Each lane represents an independent measurement. (D) Quantification of p-GSK activation in OPCs in response to treatments (one-way ANOVA, P < 0.005; *, P < 0.05 PBS vs rm-TIMP-1). (E-G) Representative images of mature OLs in primary culture (MBP+/Olig2+) following treatment with either rm-TIMP-1, MK2206, or rm-TIMP-1 with MK2206. (H) Quantification of MK2206 effect on MBP+/Olig2+ mature OLs in response to rm-TIMP-1 and effect of blocking Akt using MK2206. Data are presented as the percent mean ± SEM compared to PBS vehicle control set to 100% differentiation (one-way ANOVA, Dunnett’s multiple comparisons test: PBS vs. MK2006 [****, P < 0.0001], PBS vs. MK2206 + rm-TIMP-1 [****, P < 0.0001]). (I) Quantitative analysis of number of Olig2+ cells counted per field presented as mean ± SEM (one-way ANOVA, P = 0.1425, n.s. = not significant).

    Article Snippet: Primary antisera used were non-phosphorylated “active” β-catenin (Ser33/37/Thr41, Cell Signaling), GSK-3α/β (Cell Signaling), p-GSK-3α/β (Ser21/9, Cell Signaling), pAkt (Ser473, Cell Signaling), pan-Akt (Cell Signaling), and β-actin (Cell Signaling).

    Techniques: Western Blot, Expressing, Activation Assay, Blocking Assay

    siRNA-Mediated Silencing of GSK-3α and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells

    Journal: Molecular Therapy

    Article Title: 6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells

    doi: 10.1016/j.ymthe.2016.10.017

    Figure Lengend Snippet: siRNA-Mediated Silencing of GSK-3α and GSK-3β Suppresses AR Expression and AR Signaling in Prostate Cancer Cells

    Article Snippet: The anti-GSK-3α siRNA (6312) and the anti-GSK-3α/β-siRNA (6301, referred as si-GSK-3α/β-1) were purchased from Cell Signaling Technology, and the anti-GSK-3β siRNA (s6241) from Thermo Fisher Scientific.

    Techniques: Expressing

    GSK3 signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3 . Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.

    Journal: The Journal of Cell Biology

    Article Title: GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

    doi: 10.1083/jcb.201406020

    Figure Lengend Snippet: GSK3 signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3 . Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.

    Article Snippet: For specific knockdown of endogenous DP, two oligonucleotides targeting the 3′ UTR of DP were purchased from IDT and were used as a mix (sense sequences: 5′-AAUUAAUACCAAACCAAA-3′ and 5′-GCAGUAGAGUGAUAGGA-3′). siRNA controls used a pool of siRNA with sequences: 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, 5′-AUGAACGUGAAUUGCUCAA-3′, and 5′-UGGUUUACAUGUCGACUAA-3′. siRNA targeting GSK3 α/β isoforms were purchased from Cell Signaling Technology.

    Techniques: Staining, Immunofluorescence, Transfection, Fluorescence, Western Blot, Stable Transfection, Expressing

    GSK3 is recruited to DP to modulate DP–IF complexes. (A) Endogenous DP associates with HA-GSK3 in HA immunoprecipitations in HEK 293 cells, assessed by immunoblotting. (B) SCC9 cells were fixed, and DP and GSK3 associations were analyzed by PLA analysis using anti-DP (NW6) and anti-GSK3 antibodies. Direct interactions (red) and nuclei (DAPI, blue). Bars, 10 µm. (B′) PLA fluorescent spots are counted and divided by the total number of nuclei in an image. Data were obtained from antibody pairs for >500 cells per experiment for three independent experiments. *, P < 0.001. Error bars indicate SEM. (C and C′) Phospho-DP in LiCl- or NaCl-treated SCC9s, assessed by immunoblotting. Full-length endogenous DP has two known isoforms, DPI (310 kD) and DPII (250 kD) that are generated by alternative splicing. Both of these contain S2849 and are recognized by the phospho-DP antibody. Densitometry quantification of three independent experiments. Error bars indicate SEM. (D) DP phosphorylation in siCtr or siGSK3-treated cells, assessed by immunoblotting. (E and E′) Phospho-DP in control or caGSK3 transfected cells, and lysates were assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (F) S-tag affinity pull-down of HA-GSK3 and DP S-tag (DP CT) HEK 293 cells, assessed by immunoblotting. (G) NaCl-, LiCl-, or Ly-294,002 (GSK3 activator)–treated DP S-tag (DP CT) expressing cell lysates, assessed by immunoblotting. DP S-tag (DP CT) is present as a doublet where the lower band (32 kD) contains phosphorylation of S2849, whereas the higher band (red arrow, 36 kD) contains both phosphorylation of S2849 and downstream GSK3-phosphorylated sites (see cartoon in ). (G′) Densitometry quantification of G from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) NaCl- or LiCl-treated DP S-tag (DP CT) cells were transfected with control or caGSK3, assessed by immunoblotting. Long exposure and short exposure of DP CT blot shown to visualize upper phospho-states of DP (36 kD). (H′) Densitometry quantification of phospho-DP divided by total DP from three independent experiments. Error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

    doi: 10.1083/jcb.201406020

    Figure Lengend Snippet: GSK3 is recruited to DP to modulate DP–IF complexes. (A) Endogenous DP associates with HA-GSK3 in HA immunoprecipitations in HEK 293 cells, assessed by immunoblotting. (B) SCC9 cells were fixed, and DP and GSK3 associations were analyzed by PLA analysis using anti-DP (NW6) and anti-GSK3 antibodies. Direct interactions (red) and nuclei (DAPI, blue). Bars, 10 µm. (B′) PLA fluorescent spots are counted and divided by the total number of nuclei in an image. Data were obtained from antibody pairs for >500 cells per experiment for three independent experiments. *, P < 0.001. Error bars indicate SEM. (C and C′) Phospho-DP in LiCl- or NaCl-treated SCC9s, assessed by immunoblotting. Full-length endogenous DP has two known isoforms, DPI (310 kD) and DPII (250 kD) that are generated by alternative splicing. Both of these contain S2849 and are recognized by the phospho-DP antibody. Densitometry quantification of three independent experiments. Error bars indicate SEM. (D) DP phosphorylation in siCtr or siGSK3-treated cells, assessed by immunoblotting. (E and E′) Phospho-DP in control or caGSK3 transfected cells, and lysates were assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (F) S-tag affinity pull-down of HA-GSK3 and DP S-tag (DP CT) HEK 293 cells, assessed by immunoblotting. (G) NaCl-, LiCl-, or Ly-294,002 (GSK3 activator)–treated DP S-tag (DP CT) expressing cell lysates, assessed by immunoblotting. DP S-tag (DP CT) is present as a doublet where the lower band (32 kD) contains phosphorylation of S2849, whereas the higher band (red arrow, 36 kD) contains both phosphorylation of S2849 and downstream GSK3-phosphorylated sites (see cartoon in ). (G′) Densitometry quantification of G from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) NaCl- or LiCl-treated DP S-tag (DP CT) cells were transfected with control or caGSK3, assessed by immunoblotting. Long exposure and short exposure of DP CT blot shown to visualize upper phospho-states of DP (36 kD). (H′) Densitometry quantification of phospho-DP divided by total DP from three independent experiments. Error bars indicate SEM.

    Article Snippet: For specific knockdown of endogenous DP, two oligonucleotides targeting the 3′ UTR of DP were purchased from IDT and were used as a mix (sense sequences: 5′-AAUUAAUACCAAACCAAA-3′ and 5′-GCAGUAGAGUGAUAGGA-3′). siRNA controls used a pool of siRNA with sequences: 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, 5′-AUGAACGUGAAUUGCUCAA-3′, and 5′-UGGUUUACAUGUCGACUAA-3′. siRNA targeting GSK3 α/β isoforms were purchased from Cell Signaling Technology.

    Techniques: Western Blot, Generated, Transfection, Expressing

    GSK3 promotes phosphorylation cascades of the DP CT tail. (A) GSK3 cascade 1 (solid arrows) and 2 (dotted arrows) in WT, S2849G (red), and R2834H (*) DP. (B) NaCl- or LiCl-treated WT DP S-tag (DP CT) cell lysates were assessed by phos-tag immunoblot analyses (red arrow, 36 kD). (C and C′) Phosphorylation of DP S-tag (DP CT) point mutations, S2845A and T2853A, assessed by immunoblotting. Red arrows mark GSK3 phosphorylation of DP. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (D and D′) NaCl-, LiCl-, DMSO-, or Ly-294,002–treated WT, S2845A, or T2853A DP S-tag (DP CT) lysates, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (E and E′) DMSO-, KT 5720-, Gö 6976-, or LiCl-treated DP S-tag (DP CT) lysates, assessed by immunoblotting (red arrow, 36 kD). Densitometry quantification of three independent experiments. Error bars indicate SEM. (F) DP S-tag (DP CT) expressing HEK 293 cells were treated with kinase inhibitors targeting CKI, CKII, MNKI, atypical PKC, and DYRK kinases, assessed by immunoblotting. (G) Control, 40 mM LiCl-, and PMA-treated SCC9s, assessed by immunofluorescence. Bars, 10 µm. (H and H′) GSK3 interactions with S2849G and WT DP S-tag (DP CT) in HEK 293 cells were examined after S-tag pull-down analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting and quantified by densitometry. *, P < 0.03. Error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

    doi: 10.1083/jcb.201406020

    Figure Lengend Snippet: GSK3 promotes phosphorylation cascades of the DP CT tail. (A) GSK3 cascade 1 (solid arrows) and 2 (dotted arrows) in WT, S2849G (red), and R2834H (*) DP. (B) NaCl- or LiCl-treated WT DP S-tag (DP CT) cell lysates were assessed by phos-tag immunoblot analyses (red arrow, 36 kD). (C and C′) Phosphorylation of DP S-tag (DP CT) point mutations, S2845A and T2853A, assessed by immunoblotting. Red arrows mark GSK3 phosphorylation of DP. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (D and D′) NaCl-, LiCl-, DMSO-, or Ly-294,002–treated WT, S2845A, or T2853A DP S-tag (DP CT) lysates, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (E and E′) DMSO-, KT 5720-, Gö 6976-, or LiCl-treated DP S-tag (DP CT) lysates, assessed by immunoblotting (red arrow, 36 kD). Densitometry quantification of three independent experiments. Error bars indicate SEM. (F) DP S-tag (DP CT) expressing HEK 293 cells were treated with kinase inhibitors targeting CKI, CKII, MNKI, atypical PKC, and DYRK kinases, assessed by immunoblotting. (G) Control, 40 mM LiCl-, and PMA-treated SCC9s, assessed by immunofluorescence. Bars, 10 µm. (H and H′) GSK3 interactions with S2849G and WT DP S-tag (DP CT) in HEK 293 cells were examined after S-tag pull-down analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting and quantified by densitometry. *, P < 0.03. Error bars indicate SEM.

    Article Snippet: For specific knockdown of endogenous DP, two oligonucleotides targeting the 3′ UTR of DP were purchased from IDT and were used as a mix (sense sequences: 5′-AAUUAAUACCAAACCAAA-3′ and 5′-GCAGUAGAGUGAUAGGA-3′). siRNA controls used a pool of siRNA with sequences: 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, 5′-AUGAACGUGAAUUGCUCAA-3′, and 5′-UGGUUUACAUGUCGACUAA-3′. siRNA targeting GSK3 α/β isoforms were purchased from Cell Signaling Technology.

    Techniques: Western Blot, Expressing, Immunofluorescence

    AC mutation alters DP phosphorylation by GSK3. (A) GSK3 interactions with R2834H and WT DP S-tag (DP CT) in HEK 293 cells were examined following S-tag pulldown analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting. (A′) Densitometry quantification of GSK3 interactions with WT and R2834H DP S-tag (DP CT). *, P < 0.03. Error bars indicate SEM. (B) PLA of WT, R2834H, or S2849G DP-GFP cells incubated with primary antibody pairs of GFP-GSK3. Nuclei were stained with DAPI (blue). (B′) PLA fluorescence intensity divided by number of nuclei was performed for each cell line using primary antibody pairs of GFP-GSK3 and with controls GSK3-RIgG or GFP-MIgG. Data quantification was analyzed from three separate experiments (>500 nuclei per experiment). *, P < 0.05. Error bars indicate SEM. (C) Control or caGSK3-transfected and LiCl- or Ly-294,002 (GSK3 Act.)–treated DP-GFP cell lines, assessed by immunofluorescence. Bars, 10 µm. (D and D′) R2834H DP S-tag (DP CT) cells were treated with NaCl or LiCl or transfected with caGSK3, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

    doi: 10.1083/jcb.201406020

    Figure Lengend Snippet: AC mutation alters DP phosphorylation by GSK3. (A) GSK3 interactions with R2834H and WT DP S-tag (DP CT) in HEK 293 cells were examined following S-tag pulldown analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting. (A′) Densitometry quantification of GSK3 interactions with WT and R2834H DP S-tag (DP CT). *, P < 0.03. Error bars indicate SEM. (B) PLA of WT, R2834H, or S2849G DP-GFP cells incubated with primary antibody pairs of GFP-GSK3. Nuclei were stained with DAPI (blue). (B′) PLA fluorescence intensity divided by number of nuclei was performed for each cell line using primary antibody pairs of GFP-GSK3 and with controls GSK3-RIgG or GFP-MIgG. Data quantification was analyzed from three separate experiments (>500 nuclei per experiment). *, P < 0.05. Error bars indicate SEM. (C) Control or caGSK3-transfected and LiCl- or Ly-294,002 (GSK3 Act.)–treated DP-GFP cell lines, assessed by immunofluorescence. Bars, 10 µm. (D and D′) R2834H DP S-tag (DP CT) cells were treated with NaCl or LiCl or transfected with caGSK3, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM.

    Article Snippet: For specific knockdown of endogenous DP, two oligonucleotides targeting the 3′ UTR of DP were purchased from IDT and were used as a mix (sense sequences: 5′-AAUUAAUACCAAACCAAA-3′ and 5′-GCAGUAGAGUGAUAGGA-3′). siRNA controls used a pool of siRNA with sequences: 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, 5′-AUGAACGUGAAUUGCUCAA-3′, and 5′-UGGUUUACAUGUCGACUAA-3′. siRNA targeting GSK3 α/β isoforms were purchased from Cell Signaling Technology.

    Techniques: Mutagenesis, Western Blot, Incubation, Staining, Fluorescence, Transfection, Immunofluorescence

    GSK3 phosphorylation of DP altered in R2834H DP mice. (A) Transgenic mouse hearts were lysed in urea sample buffer and phosphorylation of WT and R2834H DP-Flag were analyzed by immunoblotting using DP phospho-specific and GSK3 substrate phospho-specific antibodies. Total levels of DP expression were assessed using anti-DP (NW6) and Flag. Tubulin was used as a loading control. (A′) Densitometry quantification indicates that although total levels of DP expression are similar, phosphorylation of R2834H DP was dramatically decreased compared with WT DP using both the DP-specific phospho-antibody and a GSK3 substrate-specific phospho-antibody. The data shown are from a single representative experiment out of three repeats ( n = 3). Error bars represent SEM. *, P < 0.001. (B) R2834H DP-trangenic mice demonstrate altered cytoplasmic localization of DP and disruption of cell–cell contacts in cardiac tissues, assessed by confocal immunofluorescence. WT and R2834H DP cardiac sections were stained with anti-Flag and anti-desmin antibodies. (C) PLA analyses were performed with WT and R2834H DP cardiac sections incubated with primary antibody pairs DP (NW6)-GSK3, DP (NW6)-MIgG, or GSK3-RIgG. Colocalization allows for hybridization, ligation, and amplification of oligonucleotide adducts fused to secondary antibodies, ultimately producing a fluorescent spot (red) in situ. Blue DAPI staining marks nuclei. Bars, 10 µm. (C′) For the quantification of PLA, PLA spots were counted and divided by total number of nuclei in a frame. Quantification shows a statistically significant enhancement of signal for the DP-GSK3 antibody pairing in WT hearts compared with R2834H transgenic mouse hearts. *, P < 0.03; **, P < 0.001; from >500 cells counted from four independent experiments. Error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

    doi: 10.1083/jcb.201406020

    Figure Lengend Snippet: GSK3 phosphorylation of DP altered in R2834H DP mice. (A) Transgenic mouse hearts were lysed in urea sample buffer and phosphorylation of WT and R2834H DP-Flag were analyzed by immunoblotting using DP phospho-specific and GSK3 substrate phospho-specific antibodies. Total levels of DP expression were assessed using anti-DP (NW6) and Flag. Tubulin was used as a loading control. (A′) Densitometry quantification indicates that although total levels of DP expression are similar, phosphorylation of R2834H DP was dramatically decreased compared with WT DP using both the DP-specific phospho-antibody and a GSK3 substrate-specific phospho-antibody. The data shown are from a single representative experiment out of three repeats ( n = 3). Error bars represent SEM. *, P < 0.001. (B) R2834H DP-trangenic mice demonstrate altered cytoplasmic localization of DP and disruption of cell–cell contacts in cardiac tissues, assessed by confocal immunofluorescence. WT and R2834H DP cardiac sections were stained with anti-Flag and anti-desmin antibodies. (C) PLA analyses were performed with WT and R2834H DP cardiac sections incubated with primary antibody pairs DP (NW6)-GSK3, DP (NW6)-MIgG, or GSK3-RIgG. Colocalization allows for hybridization, ligation, and amplification of oligonucleotide adducts fused to secondary antibodies, ultimately producing a fluorescent spot (red) in situ. Blue DAPI staining marks nuclei. Bars, 10 µm. (C′) For the quantification of PLA, PLA spots were counted and divided by total number of nuclei in a frame. Quantification shows a statistically significant enhancement of signal for the DP-GSK3 antibody pairing in WT hearts compared with R2834H transgenic mouse hearts. *, P < 0.03; **, P < 0.001; from >500 cells counted from four independent experiments. Error bars indicate SEM.

    Article Snippet: For specific knockdown of endogenous DP, two oligonucleotides targeting the 3′ UTR of DP were purchased from IDT and were used as a mix (sense sequences: 5′-AAUUAAUACCAAACCAAA-3′ and 5′-GCAGUAGAGUGAUAGGA-3′). siRNA controls used a pool of siRNA with sequences: 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, 5′-AUGAACGUGAAUUGCUCAA-3′, and 5′-UGGUUUACAUGUCGACUAA-3′. siRNA targeting GSK3 α/β isoforms were purchased from Cell Signaling Technology.

    Techniques: Transgenic Assay, Western Blot, Expressing, Immunofluorescence, Staining, Incubation, Hybridization, Ligation, Amplification, In Situ