phosphorylated  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated
    Phosphorylated, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phosphorylated  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated
    Phosphorylated, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p gsk3β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p gsk3β
    TGFβr1-mediated LPS/D-GalN–induced ALF was dependent <t>on</t> <t>GSK-3β/Nrf2</t> signaling. ( A ) Immunofluorescence co-staining of TGFβ1 and <t>GSK3β</t> from WT mice treated with vehicle or LPS/D-GalN and ( B ) quantitative assay (n = 5). Scale bars : 100 μm. ( C ) Electron microscopy of liver tissues from WT mice treated with vehicle or LPS/D-GalN. ( D ) Glucose assay of mice serum treated with vehicle or LPS/D-GalN (n = 3). ( E ) Representative pictures <t>for</t> <t>p-GSK3β</t> from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( F ) quantitative results (n = 5). Scale bars : 100 μm. ( G ) Nrf2 immunofluorescence staining of TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment (n = 5). Scale bars : 10 μm. ( H ) Representative Western blots of p-GSK3β, Nrf2 from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( I ) ratios of each protein to GAPDH (n = 3). ( J ) Immunofluorescence co-staining of p-GSK3β (red) and Nrf2 (green) from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment. Scale bars : 100 μm. All data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    P Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Hepatic TGFβr1 Deficiency Attenuates Lipopolysaccharide/D-Galactosamine–Induced Acute Liver Failure Through Inhibiting GSK3β–Nrf2–Mediated Hepatocyte Apoptosis and Ferroptosis"

    Article Title: Hepatic TGFβr1 Deficiency Attenuates Lipopolysaccharide/D-Galactosamine–Induced Acute Liver Failure Through Inhibiting GSK3β–Nrf2–Mediated Hepatocyte Apoptosis and Ferroptosis

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2022.02.009

    TGFβr1-mediated LPS/D-GalN–induced ALF was dependent on GSK-3β/Nrf2 signaling. ( A ) Immunofluorescence co-staining of TGFβ1 and GSK3β from WT mice treated with vehicle or LPS/D-GalN and ( B ) quantitative assay (n = 5). Scale bars : 100 μm. ( C ) Electron microscopy of liver tissues from WT mice treated with vehicle or LPS/D-GalN. ( D ) Glucose assay of mice serum treated with vehicle or LPS/D-GalN (n = 3). ( E ) Representative pictures for p-GSK3β from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( F ) quantitative results (n = 5). Scale bars : 100 μm. ( G ) Nrf2 immunofluorescence staining of TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment (n = 5). Scale bars : 10 μm. ( H ) Representative Western blots of p-GSK3β, Nrf2 from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( I ) ratios of each protein to GAPDH (n = 3). ( J ) Immunofluorescence co-staining of p-GSK3β (red) and Nrf2 (green) from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment. Scale bars : 100 μm. All data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: TGFβr1-mediated LPS/D-GalN–induced ALF was dependent on GSK-3β/Nrf2 signaling. ( A ) Immunofluorescence co-staining of TGFβ1 and GSK3β from WT mice treated with vehicle or LPS/D-GalN and ( B ) quantitative assay (n = 5). Scale bars : 100 μm. ( C ) Electron microscopy of liver tissues from WT mice treated with vehicle or LPS/D-GalN. ( D ) Glucose assay of mice serum treated with vehicle or LPS/D-GalN (n = 3). ( E ) Representative pictures for p-GSK3β from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( F ) quantitative results (n = 5). Scale bars : 100 μm. ( G ) Nrf2 immunofluorescence staining of TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment (n = 5). Scale bars : 10 μm. ( H ) Representative Western blots of p-GSK3β, Nrf2 from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( I ) ratios of each protein to GAPDH (n = 3). ( J ) Immunofluorescence co-staining of p-GSK3β (red) and Nrf2 (green) from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment. Scale bars : 100 μm. All data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Immunofluorescence, Staining, Electron Microscopy, Glucose Assay, Western Blot

    p gsk 3 β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p gsk 3 β
    P Gsk 3 β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p gsk3β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p gsk3β
    P Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p gsk 3 β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p gsk 3 β
    P Gsk 3 β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho gsk3 β 5558 monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho gsk3 β 5558 monoclonal antibody
    BHD activates NRF2 and promotes the phosphorylation of <t>AKT/GSK3</t> β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.
    Phospho Gsk3 β 5558 Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia"

    Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2021/1470829

    BHD activates NRF2 and promotes the phosphorylation of AKT/GSK3 β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.
    Figure Legend Snippet: BHD activates NRF2 and promotes the phosphorylation of AKT/GSK3 β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.

    Techniques Used: Expressing

    BHD promotes revascularization of hindlimb of HLI mice via AKT/GSK3 β /NRF2 signal pathway. (a) The mRNA expression of NRF2 after injection of Ad-NRF2-shRNA ( n = 4). (b, c) The tissue perfusion of hindlimb in HLI mice after 0, 7, and 14 days of BHD treatment ( n = 4). (d–f) The protein expression and quantitative analysis of NRF2, VEGFA, TNF- α , IL-1 β , IL-6, CAT, NQO-1, and HO-1 and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. NS, P > 0.05; ∗ P < 0.05, compared with HLI+BHD+sh-NRF2 group.
    Figure Legend Snippet: BHD promotes revascularization of hindlimb of HLI mice via AKT/GSK3 β /NRF2 signal pathway. (a) The mRNA expression of NRF2 after injection of Ad-NRF2-shRNA ( n = 4). (b, c) The tissue perfusion of hindlimb in HLI mice after 0, 7, and 14 days of BHD treatment ( n = 4). (d–f) The protein expression and quantitative analysis of NRF2, VEGFA, TNF- α , IL-1 β , IL-6, CAT, NQO-1, and HO-1 and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. NS, P > 0.05; ∗ P < 0.05, compared with HLI+BHD+sh-NRF2 group.

    Techniques Used: Expressing, Injection, shRNA

    BHD activates NRF2 through Akt/GSK3 β signal pathway. After injection of inhibitor, the protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+BHD group.
    Figure Legend Snippet: BHD activates NRF2 through Akt/GSK3 β signal pathway. After injection of inhibitor, the protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+BHD group.

    Techniques Used: Injection, Expressing

    anti phospho ser 9 gsk3β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho ser 9 gsk3β
    Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and <t>GSK3β</t> were increased, whereas those of p90, SOX2, Akt and p <t>Ser</t> <t>9</t> -GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.
    Anti Phospho Ser 9 Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RIP1-dependent Apoptosis and Differentiation Regulated by Skp2 and Akt/GSK3β in Acute Myeloid Leukemia"

    Article Title: RIP1-dependent Apoptosis and Differentiation Regulated by Skp2 and Akt/GSK3β in Acute Myeloid Leukemia

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.68385

    Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and GSK3β were increased, whereas those of p90, SOX2, Akt and p Ser 9 -GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.
    Figure Legend Snippet: Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and GSK3β were increased, whereas those of p90, SOX2, Akt and p Ser 9 -GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.

    Techniques Used: Inhibition, Expressing, CCK-8 Assay, Flow Cytometry, Staining, Transfection, Western Blot

    Inhibition of the RIP1 protein negatively regulates the Akt/GSK3β pathway and GSK3β dephosphorylation suppresses RIP1 expression. (A) NB4 cells with control or Skp2 knockdown, treated with or without Nec-1, harvested after 48h, followed by Western blot analysis. (B) An endogenous IP assay demonstrating that the knockdown of Skp2 resulted in a reduction of GSK3β in both K48- and K63-linked polyubiquitination of U937 cells. (C) C/EBPβ mRNA levels in U937 cells treated with SB216763 alone or in combination with ATRA (10 -6 M) determined by RT-qPCR. (D) The effects of the GSK3β activation concerning ATRA-induced differentiation in U937 cells. U937 cells were treated with ATRA, SB216763, or a combination of these agents. Cell extracts were prepared and the levels of the indicated proteins were detected by immunoblotting. (E) Immunoblotting and quantification of protein levels upon treatment with SB216763 (GSK3β inhibitor) at the indicated times in various AML cell lines. Data presented as the mean ±SD of three independent experiments. P values were determined by Student's t-test. *p< 0.05, **p< 0.01, ***p< 0.001.
    Figure Legend Snippet: Inhibition of the RIP1 protein negatively regulates the Akt/GSK3β pathway and GSK3β dephosphorylation suppresses RIP1 expression. (A) NB4 cells with control or Skp2 knockdown, treated with or without Nec-1, harvested after 48h, followed by Western blot analysis. (B) An endogenous IP assay demonstrating that the knockdown of Skp2 resulted in a reduction of GSK3β in both K48- and K63-linked polyubiquitination of U937 cells. (C) C/EBPβ mRNA levels in U937 cells treated with SB216763 alone or in combination with ATRA (10 -6 M) determined by RT-qPCR. (D) The effects of the GSK3β activation concerning ATRA-induced differentiation in U937 cells. U937 cells were treated with ATRA, SB216763, or a combination of these agents. Cell extracts were prepared and the levels of the indicated proteins were detected by immunoblotting. (E) Immunoblotting and quantification of protein levels upon treatment with SB216763 (GSK3β inhibitor) at the indicated times in various AML cell lines. Data presented as the mean ±SD of three independent experiments. P values were determined by Student's t-test. *p< 0.05, **p< 0.01, ***p< 0.001.

    Techniques Used: Inhibition, De-Phosphorylation Assay, Expressing, Western Blot, Quantitative RT-PCR, Activation Assay

    rabbit anti p gsk3β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti p gsk3β
    The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total <t>β-catenin,</t> <t>GSK-3β,</t> <t>and</t> <t>p-GSK-3β</t> were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.
    Rabbit Anti P Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells"

    Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

    Journal: Theranostics

    doi: 10.7150/thno.35914

    The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total β-catenin, GSK-3β, and p-GSK-3β were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.
    Figure Legend Snippet: The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total β-catenin, GSK-3β, and p-GSK-3β were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.

    Techniques Used: Cell Culture, Western Blot, Expressing, Staining

    thr197 phosphorylation level  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc thr197 phosphorylation level
    LGR5 expression level was correlated with PKA activation in human breast cancer cells. ( A, B ) Phosphorylation at <t>Thr197</t> on the PKA catalytic subunits α and β (PRKACs) was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. Western blot analysis was performed using the same method as in . ( C ) Representative result of Western blot evaluating the protein expression level and Thr197 phosphorylation level of PKA catalytic subunits α and β in each group of cells. ( D ) PKA kinase activity was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. The t test was used for significance test. * p<0.05.
    Thr197 Phosphorylation Level, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates β-Catenin Signaling in Breast Cancer Cells via Protein Kinase A"

    Article Title: G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates β-Catenin Signaling in Breast Cancer Cells via Protein Kinase A

    Journal: Medical Science Monitor Basic Research

    doi: 10.12659/MSMBR.912411

    LGR5 expression level was correlated with PKA activation in human breast cancer cells. ( A, B ) Phosphorylation at Thr197 on the PKA catalytic subunits α and β (PRKACs) was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. Western blot analysis was performed using the same method as in . ( C ) Representative result of Western blot evaluating the protein expression level and Thr197 phosphorylation level of PKA catalytic subunits α and β in each group of cells. ( D ) PKA kinase activity was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. The t test was used for significance test. * p<0.05.
    Figure Legend Snippet: LGR5 expression level was correlated with PKA activation in human breast cancer cells. ( A, B ) Phosphorylation at Thr197 on the PKA catalytic subunits α and β (PRKACs) was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. Western blot analysis was performed using the same method as in . ( C ) Representative result of Western blot evaluating the protein expression level and Thr197 phosphorylation level of PKA catalytic subunits α and β in each group of cells. ( D ) PKA kinase activity was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. The t test was used for significance test. * p<0.05.

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

    anti phospho gsk3 β ser 9  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti phospho gsk3 β ser 9
    Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and <t>GSK3</t> β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).
    Anti Phospho Gsk3 β Ser 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway"

    Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/8597897

    Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and GSK3 β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).
    Figure Legend Snippet: Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and GSK3 β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).

    Techniques Used: Activation Assay, In Vivo, Western Blot

    Salidroside (SAL) improves insulin sensitivity and suppresses NLRP3 inflammasome activation in hepatocytes. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), primary mouse hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, and 10 μ M) for 72 h. For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. Protein sample were extracted from hepatocytes or supernatant (SN). The phosphorylation of Akt and GSK3 β (a, d) and the activation of NLRP3 inflammasome (c, f) were analyzed by immunoblot. The supernatant IL-1 β concentration (b, e) was measured by ELISA method. † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a), (c), (d), and (f): n = 4; (b) and (e): n = 3).
    Figure Legend Snippet: Salidroside (SAL) improves insulin sensitivity and suppresses NLRP3 inflammasome activation in hepatocytes. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), primary mouse hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, and 10 μ M) for 72 h. For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. Protein sample were extracted from hepatocytes or supernatant (SN). The phosphorylation of Akt and GSK3 β (a, d) and the activation of NLRP3 inflammasome (c, f) were analyzed by immunoblot. The supernatant IL-1 β concentration (b, e) was measured by ELISA method. † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a), (c), (d), and (f): n = 4; (b) and (e): n = 3).

    Techniques Used: Activation Assay, Cell Culture, Incubation, In Vitro, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Salidroside (SAL) protects hepatocytes against ROS overproduction. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, 10 μ M) and N-acetylcysteine (NAC, 5 mM) for 72 h. The ROS levels were detected using DCF-DA and MitoSOX as indicated (a–d). The Oil Red O staining (e) and lipid content analysis (f) were performed. Scale bar = 200 μ m. Protein sample was extracted from hepatocytes or supernatant (SN). For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. The phosphorylation of AMPK, ACC (g), Akt, and GSK3 β (h) and the activation of NLRP3 inflammasome (j) were analyzed by immunoblot. The supernatant IL-1 β concentration was measured by ELISA method (i). † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a)–(d), (f)–(h), and (j): n = 4; (e) and (i): n = 3).
    Figure Legend Snippet: Salidroside (SAL) protects hepatocytes against ROS overproduction. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, 10 μ M) and N-acetylcysteine (NAC, 5 mM) for 72 h. The ROS levels were detected using DCF-DA and MitoSOX as indicated (a–d). The Oil Red O staining (e) and lipid content analysis (f) were performed. Scale bar = 200 μ m. Protein sample was extracted from hepatocytes or supernatant (SN). For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. The phosphorylation of AMPK, ACC (g), Akt, and GSK3 β (h) and the activation of NLRP3 inflammasome (j) were analyzed by immunoblot. The supernatant IL-1 β concentration was measured by ELISA method (i). † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a)–(d), (f)–(h), and (j): n = 4; (e) and (i): n = 3).

    Techniques Used: Cell Culture, Incubation, Staining, In Vitro, Activation Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

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    Cell Signaling Technology Inc phosphorylated
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    TGFβr1-mediated LPS/D-GalN–induced ALF was dependent <t>on</t> <t>GSK-3β/Nrf2</t> signaling. ( A ) Immunofluorescence co-staining of TGFβ1 and <t>GSK3β</t> from WT mice treated with vehicle or LPS/D-GalN and ( B ) quantitative assay (n = 5). Scale bars : 100 μm. ( C ) Electron microscopy of liver tissues from WT mice treated with vehicle or LPS/D-GalN. ( D ) Glucose assay of mice serum treated with vehicle or LPS/D-GalN (n = 3). ( E ) Representative pictures <t>for</t> <t>p-GSK3β</t> from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( F ) quantitative results (n = 5). Scale bars : 100 μm. ( G ) Nrf2 immunofluorescence staining of TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment (n = 5). Scale bars : 10 μm. ( H ) Representative Western blots of p-GSK3β, Nrf2 from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( I ) ratios of each protein to GAPDH (n = 3). ( J ) Immunofluorescence co-staining of p-GSK3β (red) and Nrf2 (green) from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment. Scale bars : 100 μm. All data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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    Cell Signaling Technology Inc phospho gsk3 β 5558 monoclonal antibody
    BHD activates NRF2 and promotes the phosphorylation of <t>AKT/GSK3</t> β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.
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    Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and <t>GSK3β</t> were increased, whereas those of p90, SOX2, Akt and p <t>Ser</t> <t>9</t> -GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.
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    The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total <t>β-catenin,</t> <t>GSK-3β,</t> <t>and</t> <t>p-GSK-3β</t> were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.
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    LGR5 expression level was correlated with PKA activation in human breast cancer cells. ( A, B ) Phosphorylation at <t>Thr197</t> on the PKA catalytic subunits α and β (PRKACs) was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. Western blot analysis was performed using the same method as in . ( C ) Representative result of Western blot evaluating the protein expression level and Thr197 phosphorylation level of PKA catalytic subunits α and β in each group of cells. ( D ) PKA kinase activity was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. The t test was used for significance test. * p<0.05.
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    Cell Signaling Technology Inc anti phospho gsk3 β ser 9
    Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and <t>GSK3</t> β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).
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    Image Search Results


    TGFβr1-mediated LPS/D-GalN–induced ALF was dependent on GSK-3β/Nrf2 signaling. ( A ) Immunofluorescence co-staining of TGFβ1 and GSK3β from WT mice treated with vehicle or LPS/D-GalN and ( B ) quantitative assay (n = 5). Scale bars : 100 μm. ( C ) Electron microscopy of liver tissues from WT mice treated with vehicle or LPS/D-GalN. ( D ) Glucose assay of mice serum treated with vehicle or LPS/D-GalN (n = 3). ( E ) Representative pictures for p-GSK3β from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( F ) quantitative results (n = 5). Scale bars : 100 μm. ( G ) Nrf2 immunofluorescence staining of TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment (n = 5). Scale bars : 10 μm. ( H ) Representative Western blots of p-GSK3β, Nrf2 from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( I ) ratios of each protein to GAPDH (n = 3). ( J ) Immunofluorescence co-staining of p-GSK3β (red) and Nrf2 (green) from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment. Scale bars : 100 μm. All data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Hepatic TGFβr1 Deficiency Attenuates Lipopolysaccharide/D-Galactosamine–Induced Acute Liver Failure Through Inhibiting GSK3β–Nrf2–Mediated Hepatocyte Apoptosis and Ferroptosis

    doi: 10.1016/j.jcmgh.2022.02.009

    Figure Lengend Snippet: TGFβr1-mediated LPS/D-GalN–induced ALF was dependent on GSK-3β/Nrf2 signaling. ( A ) Immunofluorescence co-staining of TGFβ1 and GSK3β from WT mice treated with vehicle or LPS/D-GalN and ( B ) quantitative assay (n = 5). Scale bars : 100 μm. ( C ) Electron microscopy of liver tissues from WT mice treated with vehicle or LPS/D-GalN. ( D ) Glucose assay of mice serum treated with vehicle or LPS/D-GalN (n = 3). ( E ) Representative pictures for p-GSK3β from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( F ) quantitative results (n = 5). Scale bars : 100 μm. ( G ) Nrf2 immunofluorescence staining of TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment (n = 5). Scale bars : 10 μm. ( H ) Representative Western blots of p-GSK3β, Nrf2 from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment and ( I ) ratios of each protein to GAPDH (n = 3). ( J ) Immunofluorescence co-staining of p-GSK3β (red) and Nrf2 (green) from TGFβr1 Δhep-CKO and TGFβr1 Δhep mice after vehicle or LPS/D-GalN treatment. Scale bars : 100 μm. All data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Article Snippet: Finally, frozen liver sections were stained with CD68 (ab201340; Abcam, Cambridge, UK), CD11b (ab52478; Abcam), Ptgs2 (ab15191; Abcam), DMT1 (ab55812; Abcam), GPX4 (ab125066; Abcam), F4/80 (DF2789; Affinity, Jiangsu, China), TGFβ1 (ER31210; huabio, Hangzhou, China; 21898-1-AP; Proteintech), Ki67 (9129s; Cell Signaling Technology, Boston, MA), GSK3β (9832s; Cell Signaling Technology), P-GSK3β (5558p; Cell Signaling Technology), Nrf2 (16396-1; Proteintech, Chicago, IL), and iNOS (ab178945; Abcam) overnight at 4°C.

    Techniques: Immunofluorescence, Staining, Electron Microscopy, Glucose Assay, Western Blot

    BHD activates NRF2 and promotes the phosphorylation of AKT/GSK3 β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

    doi: 10.1155/2021/1470829

    Figure Lengend Snippet: BHD activates NRF2 and promotes the phosphorylation of AKT/GSK3 β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.

    Article Snippet: BHD consists of seven herbal medicines which include Huang Qi, Dang Gui, Chi Shao, Chuan Xiong, Tao Ren, Hong Hua, and Di Long, with the ratio of 120 : 6 : 5 : 3 : 3 : 3 : 3, and the specific formulation was described previously [ ]; CD31 polyclonal antibody (ab28364) and VEGF polyclonal antibody (ab46154) were purchased from Abcam (UK); NAD(P)H dehydrogenase quinone 1 (NQO-1) polyclonal antibody (DF6437) was purchased from Affinity Biosciences (USA); heme oxygenase-1 (HO-1) polyclonal antibody (10701-1-AP), catalase polyclonal antibody (21260-1-AP), and histone H3 (17168-1-AP) were purchased from Proteintech (CHN); NRF2 monoclonal antibody (12721), Akt (4691) and phospho-Akt monoclonal antibody (4060), glycogen synthase kinase-3 beta (GSK3 β ) (12456) and phospho-GSK3 β (5558) monoclonal antibody, interleukin-6 (IL-6) monoclonal antibody (12912), interleukin-1beta (IL-1 β ) monoclonal antibody (12242), tumor necrosis factor- (TNF-) α monoclonal antibody (11948), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (5174) were purchased from Cell Signaling Technology (USA).

    Techniques: Expressing

    BHD promotes revascularization of hindlimb of HLI mice via AKT/GSK3 β /NRF2 signal pathway. (a) The mRNA expression of NRF2 after injection of Ad-NRF2-shRNA ( n = 4). (b, c) The tissue perfusion of hindlimb in HLI mice after 0, 7, and 14 days of BHD treatment ( n = 4). (d–f) The protein expression and quantitative analysis of NRF2, VEGFA, TNF- α , IL-1 β , IL-6, CAT, NQO-1, and HO-1 and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. NS, P > 0.05; ∗ P < 0.05, compared with HLI+BHD+sh-NRF2 group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

    doi: 10.1155/2021/1470829

    Figure Lengend Snippet: BHD promotes revascularization of hindlimb of HLI mice via AKT/GSK3 β /NRF2 signal pathway. (a) The mRNA expression of NRF2 after injection of Ad-NRF2-shRNA ( n = 4). (b, c) The tissue perfusion of hindlimb in HLI mice after 0, 7, and 14 days of BHD treatment ( n = 4). (d–f) The protein expression and quantitative analysis of NRF2, VEGFA, TNF- α , IL-1 β , IL-6, CAT, NQO-1, and HO-1 and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. NS, P > 0.05; ∗ P < 0.05, compared with HLI+BHD+sh-NRF2 group.

    Article Snippet: BHD consists of seven herbal medicines which include Huang Qi, Dang Gui, Chi Shao, Chuan Xiong, Tao Ren, Hong Hua, and Di Long, with the ratio of 120 : 6 : 5 : 3 : 3 : 3 : 3, and the specific formulation was described previously [ ]; CD31 polyclonal antibody (ab28364) and VEGF polyclonal antibody (ab46154) were purchased from Abcam (UK); NAD(P)H dehydrogenase quinone 1 (NQO-1) polyclonal antibody (DF6437) was purchased from Affinity Biosciences (USA); heme oxygenase-1 (HO-1) polyclonal antibody (10701-1-AP), catalase polyclonal antibody (21260-1-AP), and histone H3 (17168-1-AP) were purchased from Proteintech (CHN); NRF2 monoclonal antibody (12721), Akt (4691) and phospho-Akt monoclonal antibody (4060), glycogen synthase kinase-3 beta (GSK3 β ) (12456) and phospho-GSK3 β (5558) monoclonal antibody, interleukin-6 (IL-6) monoclonal antibody (12912), interleukin-1beta (IL-1 β ) monoclonal antibody (12242), tumor necrosis factor- (TNF-) α monoclonal antibody (11948), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (5174) were purchased from Cell Signaling Technology (USA).

    Techniques: Expressing, Injection, shRNA

    BHD activates NRF2 through Akt/GSK3 β signal pathway. After injection of inhibitor, the protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+BHD group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

    doi: 10.1155/2021/1470829

    Figure Lengend Snippet: BHD activates NRF2 through Akt/GSK3 β signal pathway. After injection of inhibitor, the protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+BHD group.

    Article Snippet: BHD consists of seven herbal medicines which include Huang Qi, Dang Gui, Chi Shao, Chuan Xiong, Tao Ren, Hong Hua, and Di Long, with the ratio of 120 : 6 : 5 : 3 : 3 : 3 : 3, and the specific formulation was described previously [ ]; CD31 polyclonal antibody (ab28364) and VEGF polyclonal antibody (ab46154) were purchased from Abcam (UK); NAD(P)H dehydrogenase quinone 1 (NQO-1) polyclonal antibody (DF6437) was purchased from Affinity Biosciences (USA); heme oxygenase-1 (HO-1) polyclonal antibody (10701-1-AP), catalase polyclonal antibody (21260-1-AP), and histone H3 (17168-1-AP) were purchased from Proteintech (CHN); NRF2 monoclonal antibody (12721), Akt (4691) and phospho-Akt monoclonal antibody (4060), glycogen synthase kinase-3 beta (GSK3 β ) (12456) and phospho-GSK3 β (5558) monoclonal antibody, interleukin-6 (IL-6) monoclonal antibody (12912), interleukin-1beta (IL-1 β ) monoclonal antibody (12242), tumor necrosis factor- (TNF-) α monoclonal antibody (11948), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (5174) were purchased from Cell Signaling Technology (USA).

    Techniques: Injection, Expressing

    Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and GSK3β were increased, whereas those of p90, SOX2, Akt and p Ser 9 -GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.

    Journal: International Journal of Medical Sciences

    Article Title: RIP1-dependent Apoptosis and Differentiation Regulated by Skp2 and Akt/GSK3β in Acute Myeloid Leukemia

    doi: 10.7150/ijms.68385

    Figure Lengend Snippet: Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and GSK3β were increased, whereas those of p90, SOX2, Akt and p Ser 9 -GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.

    Article Snippet: The following specific primary antibodies were used: anti-RIP1 (Cell Signaling Technology, #73271), anti-Skp2 (Santa Cruze Biotechnology, sc-74477), anti-p53 (Cell Signaling Technology, #2527), anti-p90 (Cell Signaling Technology, #9355), anti-SOX2 (Cell Signaling Technology, #3579), anti-Akt (Abcam, ab17463), anti-GSK3β (Cell Signaling Technology, #12456), anti-phospho ser 9 -GSK3β (Cell Signaling Technology, #5558), anti-Bcl2 (Abcam, ab32121), anti- ubiquitin (Santa Cruze Biotechnology, sc-8017), anti-K63-linkage specific polyubiquitin (Cell Signaling Technology, #5621),anti-K48-linkage specific polyubiquitin (Cell Signaling Technology, #8081), anti-c-Myc (Abcam, ab32072), anti-RARα (Santa Cruze Biotechnology, sc-515796), anti-C/EBPα (Cell Signaling Technology, #8178), anti-C/EBPβ (Cell Signaling Technology, #3082), anti-H3 (Bioss, no.33942M), anti-GAPDH (Cell Signaling Technology, #5174), and anti-β-actin (Boster, no.BM0627).

    Techniques: Inhibition, Expressing, CCK-8 Assay, Flow Cytometry, Staining, Transfection, Western Blot

    Inhibition of the RIP1 protein negatively regulates the Akt/GSK3β pathway and GSK3β dephosphorylation suppresses RIP1 expression. (A) NB4 cells with control or Skp2 knockdown, treated with or without Nec-1, harvested after 48h, followed by Western blot analysis. (B) An endogenous IP assay demonstrating that the knockdown of Skp2 resulted in a reduction of GSK3β in both K48- and K63-linked polyubiquitination of U937 cells. (C) C/EBPβ mRNA levels in U937 cells treated with SB216763 alone or in combination with ATRA (10 -6 M) determined by RT-qPCR. (D) The effects of the GSK3β activation concerning ATRA-induced differentiation in U937 cells. U937 cells were treated with ATRA, SB216763, or a combination of these agents. Cell extracts were prepared and the levels of the indicated proteins were detected by immunoblotting. (E) Immunoblotting and quantification of protein levels upon treatment with SB216763 (GSK3β inhibitor) at the indicated times in various AML cell lines. Data presented as the mean ±SD of three independent experiments. P values were determined by Student's t-test. *p< 0.05, **p< 0.01, ***p< 0.001.

    Journal: International Journal of Medical Sciences

    Article Title: RIP1-dependent Apoptosis and Differentiation Regulated by Skp2 and Akt/GSK3β in Acute Myeloid Leukemia

    doi: 10.7150/ijms.68385

    Figure Lengend Snippet: Inhibition of the RIP1 protein negatively regulates the Akt/GSK3β pathway and GSK3β dephosphorylation suppresses RIP1 expression. (A) NB4 cells with control or Skp2 knockdown, treated with or without Nec-1, harvested after 48h, followed by Western blot analysis. (B) An endogenous IP assay demonstrating that the knockdown of Skp2 resulted in a reduction of GSK3β in both K48- and K63-linked polyubiquitination of U937 cells. (C) C/EBPβ mRNA levels in U937 cells treated with SB216763 alone or in combination with ATRA (10 -6 M) determined by RT-qPCR. (D) The effects of the GSK3β activation concerning ATRA-induced differentiation in U937 cells. U937 cells were treated with ATRA, SB216763, or a combination of these agents. Cell extracts were prepared and the levels of the indicated proteins were detected by immunoblotting. (E) Immunoblotting and quantification of protein levels upon treatment with SB216763 (GSK3β inhibitor) at the indicated times in various AML cell lines. Data presented as the mean ±SD of three independent experiments. P values were determined by Student's t-test. *p< 0.05, **p< 0.01, ***p< 0.001.

    Article Snippet: The following specific primary antibodies were used: anti-RIP1 (Cell Signaling Technology, #73271), anti-Skp2 (Santa Cruze Biotechnology, sc-74477), anti-p53 (Cell Signaling Technology, #2527), anti-p90 (Cell Signaling Technology, #9355), anti-SOX2 (Cell Signaling Technology, #3579), anti-Akt (Abcam, ab17463), anti-GSK3β (Cell Signaling Technology, #12456), anti-phospho ser 9 -GSK3β (Cell Signaling Technology, #5558), anti-Bcl2 (Abcam, ab32121), anti- ubiquitin (Santa Cruze Biotechnology, sc-8017), anti-K63-linkage specific polyubiquitin (Cell Signaling Technology, #5621),anti-K48-linkage specific polyubiquitin (Cell Signaling Technology, #8081), anti-c-Myc (Abcam, ab32072), anti-RARα (Santa Cruze Biotechnology, sc-515796), anti-C/EBPα (Cell Signaling Technology, #8178), anti-C/EBPβ (Cell Signaling Technology, #3082), anti-H3 (Bioss, no.33942M), anti-GAPDH (Cell Signaling Technology, #5174), and anti-β-actin (Boster, no.BM0627).

    Techniques: Inhibition, De-Phosphorylation Assay, Expressing, Western Blot, Quantitative RT-PCR, Activation Assay

    The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total β-catenin, GSK-3β, and p-GSK-3β were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.

    Journal: Theranostics

    Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

    doi: 10.7150/thno.35914

    Figure Lengend Snippet: The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total β-catenin, GSK-3β, and p-GSK-3β were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.

    Article Snippet: The primary antibodies for the total proteins included: rabbit anti-Col-IV (1:500; Novus, NBP1-19632), rabbit anti-ALP (1:1000; Abcam, ab108337), goat anti-Runx2 (1:200; Santa Cruz Biotechnology, sc8566), rabbit anti-Col-I (1:1000; Abcam, ab34710), rabbit anti-Sox-2 (1:500; Proteintech, 11064-1-AP), rabbit anti-Nanog (1:500; Proteintech, 14295-1-AP), rabbit anti-Oct-4 (1:500; Cell Signaling Technology, 2750), rabbit anti-Ki67 (1:1000; Abcam, ab16667), rabbit anti-Cyclin B1 (1:500; Proteintech, 55004-1-AP), rabbit anti-Cyclin D1 (1:200; Santa Cruz Biotechnology, sc8396), rabbit anti-β-catenin (1:500; Cell Signaling Technology, 8480), rabbit anti-GSK3β (1:500; Cell Signaling Technology,12456), and rabbit anti-p-GSK3β (1:500; Cell Signaling Technology, 5558).

    Techniques: Cell Culture, Western Blot, Expressing, Staining

    LGR5 expression level was correlated with PKA activation in human breast cancer cells. ( A, B ) Phosphorylation at Thr197 on the PKA catalytic subunits α and β (PRKACs) was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. Western blot analysis was performed using the same method as in . ( C ) Representative result of Western blot evaluating the protein expression level and Thr197 phosphorylation level of PKA catalytic subunits α and β in each group of cells. ( D ) PKA kinase activity was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. The t test was used for significance test. * p<0.05.

    Journal: Medical Science Monitor Basic Research

    Article Title: G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates β-Catenin Signaling in Breast Cancer Cells via Protein Kinase A

    doi: 10.12659/MSMBR.912411

    Figure Lengend Snippet: LGR5 expression level was correlated with PKA activation in human breast cancer cells. ( A, B ) Phosphorylation at Thr197 on the PKA catalytic subunits α and β (PRKACs) was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. Western blot analysis was performed using the same method as in . ( C ) Representative result of Western blot evaluating the protein expression level and Thr197 phosphorylation level of PKA catalytic subunits α and β in each group of cells. ( D ) PKA kinase activity was increased in LGR5-overexpressing MCF-7 cells and decreased in LGR5-knockdown MDA-MB-453 cells, compared to their wild-type counterparts. The t test was used for significance test. * p<0.05.

    Article Snippet: Antibodies for detecting the protein expression level (E247, 3D10) and Thr197 phosphorylation level (D8E11, D85E12) of β-catenin and GSK-3β were purchased from Cell Signaling (Danvers, USA).

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

    Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and GSK3 β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

    doi: 10.1155/2018/8597897

    Figure Lengend Snippet: Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and GSK3 β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).

    Article Snippet: The following primary antibodies were used: anti-IL-1 β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr 172 (number 2535), anti-phospho-GSK3 β Ser 9 (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser 79 (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser 473 (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3 β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA).

    Techniques: Activation Assay, In Vivo, Western Blot

    Salidroside (SAL) improves insulin sensitivity and suppresses NLRP3 inflammasome activation in hepatocytes. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), primary mouse hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, and 10 μ M) for 72 h. For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. Protein sample were extracted from hepatocytes or supernatant (SN). The phosphorylation of Akt and GSK3 β (a, d) and the activation of NLRP3 inflammasome (c, f) were analyzed by immunoblot. The supernatant IL-1 β concentration (b, e) was measured by ELISA method. † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a), (c), (d), and (f): n = 4; (b) and (e): n = 3).

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

    doi: 10.1155/2018/8597897

    Figure Lengend Snippet: Salidroside (SAL) improves insulin sensitivity and suppresses NLRP3 inflammasome activation in hepatocytes. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), primary mouse hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, and 10 μ M) for 72 h. For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. Protein sample were extracted from hepatocytes or supernatant (SN). The phosphorylation of Akt and GSK3 β (a, d) and the activation of NLRP3 inflammasome (c, f) were analyzed by immunoblot. The supernatant IL-1 β concentration (b, e) was measured by ELISA method. † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a), (c), (d), and (f): n = 4; (b) and (e): n = 3).

    Article Snippet: The following primary antibodies were used: anti-IL-1 β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr 172 (number 2535), anti-phospho-GSK3 β Ser 9 (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser 79 (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser 473 (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3 β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA).

    Techniques: Activation Assay, Cell Culture, Incubation, In Vitro, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Salidroside (SAL) protects hepatocytes against ROS overproduction. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, 10 μ M) and N-acetylcysteine (NAC, 5 mM) for 72 h. The ROS levels were detected using DCF-DA and MitoSOX as indicated (a–d). The Oil Red O staining (e) and lipid content analysis (f) were performed. Scale bar = 200 μ m. Protein sample was extracted from hepatocytes or supernatant (SN). For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. The phosphorylation of AMPK, ACC (g), Akt, and GSK3 β (h) and the activation of NLRP3 inflammasome (j) were analyzed by immunoblot. The supernatant IL-1 β concentration was measured by ELISA method (i). † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a)–(d), (f)–(h), and (j): n = 4; (e) and (i): n = 3).

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

    doi: 10.1155/2018/8597897

    Figure Lengend Snippet: Salidroside (SAL) protects hepatocytes against ROS overproduction. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, 10 μ M) and N-acetylcysteine (NAC, 5 mM) for 72 h. The ROS levels were detected using DCF-DA and MitoSOX as indicated (a–d). The Oil Red O staining (e) and lipid content analysis (f) were performed. Scale bar = 200 μ m. Protein sample was extracted from hepatocytes or supernatant (SN). For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. The phosphorylation of AMPK, ACC (g), Akt, and GSK3 β (h) and the activation of NLRP3 inflammasome (j) were analyzed by immunoblot. The supernatant IL-1 β concentration was measured by ELISA method (i). † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a)–(d), (f)–(h), and (j): n = 4; (e) and (i): n = 3).

    Article Snippet: The following primary antibodies were used: anti-IL-1 β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr 172 (number 2535), anti-phospho-GSK3 β Ser 9 (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser 79 (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser 473 (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3 β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA).

    Techniques: Cell Culture, Incubation, Staining, In Vitro, Activation Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay