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  • 99
    Millipore mg132 mg132
    Depletion of Smurf2 accelerates degradation of CNKSR2. a Serum starved HEK293 cells were treated with <t>MG132</t> at the indicated doses and time intervals and expression of CNKSR2 was assessed using western blot indicating proteasome mediated degradation of CNKSR2. b Decline in CNKSR2 expression induced by Smurf2 siRNA was rescued by treating the cells for 4 h with 10 μM MG132 44 h post-transfection. c Depletion of Smurf2 results in enhanced polyubiquitination and proteasomal degradation of CNKSR2. CNKSR2 was immunoprecipitated from MDA-MB-231 cells transfected with Smurf2 siRNA and control siRNA. Cells were treated for 4 h with 10 μM MG132 44 h post-transfection. The corresponding lysates were denatured and immunoprecipitated (IP) with rabbit CNKSR2 antibody, followed by Western blotting (IB) of the immune complexes with a mouse anti-ubiquitin antibody. The input proteins in cell lysates were also probed by the indicated antibodies
    Mg132 Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 23 article reviews
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    94
    InvivoGen mg132
    THOC7 is involved in the (mitochondrial antiviral signaling (MAVS) signalosome and drives proteasomal degradation of TBK1. ( A ) THOC7 was involved in forming the MAVS signaling complex. 293T cells were seeded into 100-mm dishes and transfected with Flag-tagged THOC7 or empty control plasmids (10 μg) and HA-tagged RLR signaling molecules (10 μg) as indicated. After 12 h of transfection, the cells were treated with or without SeV for 12 h. Co-immunoprecipitation and immunoblot analysis was performed with the indicated antibodies. ( B ) THOC7 promoted degradation of exogenous and endogenous TBK1 in a dose-dependent manner. 293T cells were seeded into 6-well plates and transfected with increasing amounts of Flag-THOC7 (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg), and HA-TBK1 (2 μg). Twenty-four hours after transfection, immunoblot analysis was performed with the indicated antibodies. ( C ) <t>MG132</t> restored the TBK1 protein level in the presence of THOC7. 293T cells were seeded into 6-well plates and transfected with Flag-THOC7 (2 μg) and HA-TBK1 (1.5 μg). MG132 and CHX were added after 6 h of transfection and the cells were harvested for analysis after 24 h. ( D , E ) THOC7 increased the K48-linked ubiquitination of TBK1. 293T cells were seeded into 100-mm dishes and transfected with the indicated plasmids (8 μg each). Similar co-immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Quantification of western blotting bands ( A , C , E , F ) from three independent experiments was performed with Image J software. Error bars indicate SD. n = 3. *, p
    Mg132, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore mg13 2
    THOC7 is involved in the (mitochondrial antiviral signaling (MAVS) signalosome and drives proteasomal degradation of TBK1. ( A ) THOC7 was involved in forming the MAVS signaling complex. 293T cells were seeded into 100-mm dishes and transfected with Flag-tagged THOC7 or empty control plasmids (10 μg) and HA-tagged RLR signaling molecules (10 μg) as indicated. After 12 h of transfection, the cells were treated with or without SeV for 12 h. Co-immunoprecipitation and immunoblot analysis was performed with the indicated antibodies. ( B ) THOC7 promoted degradation of exogenous and endogenous TBK1 in a dose-dependent manner. 293T cells were seeded into 6-well plates and transfected with increasing amounts of Flag-THOC7 (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg), and HA-TBK1 (2 μg). Twenty-four hours after transfection, immunoblot analysis was performed with the indicated antibodies. ( C ) <t>MG132</t> restored the TBK1 protein level in the presence of THOC7. 293T cells were seeded into 6-well plates and transfected with Flag-THOC7 (2 μg) and HA-TBK1 (1.5 μg). MG132 and CHX were added after 6 h of transfection and the cells were harvested for analysis after 24 h. ( D , E ) THOC7 increased the K48-linked ubiquitination of TBK1. 293T cells were seeded into 100-mm dishes and transfected with the indicated plasmids (8 μg each). Similar co-immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Quantification of western blotting bands ( A , C , E , F ) from three independent experiments was performed with Image J software. Error bars indicate SD. n = 3. *, p
    Mg13 2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mg13 2/product/Millipore
    Average 99 stars, based on 1 article reviews
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    99
    Selleck Chemicals mg132
    ERK interacts with FBW7. (A , B) Immunoblot (IB) analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) from 293T cells transfected with Flag-WT-FBW7, or HA-ERK1 or together. Thirty hours post-transfection, cells were pretreated with 15 μM <t>MG132</t> for 10 h before harvesting. (C , D) Immunoblot analysis of WCL and immunoprecipitates from the indicated SW1990 cells stably expressing the Flag-ERK1 construct. Cells were pretreated with 15 μM MG132 for 10 h before harvesting. (E) SW1990 cell extracts were immunoprecipitated with an antibody against FBW7 or control IgG and analyzed by immunoblot analysis.
    Mg132, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc mg132
    Ubiquitin occupancy, non-ubiquitin occupied and total protein ratios were generated for all partially ubiquitinated peptides detected in <t>MG132</t> and DMSO control treated samples. a Relative ubiquitinated, non-ubiquitinated and protein ratios for all partially ubiquitinated peptides in the MG132 treated vs. native state. b Percent ubiquitin occupancy for partially ubiquitinated peptides in the MG132 treated and native conditions. c Relative ubiquitinated, non-ubiquitinated and protein ratios for all partially ubiquitinated peptides in the DMSO treated vs. native state. d Percent ubiquitin occupancy for partially ubiquitinated peptides in the DMSO treated and native conditions
    Mg132, 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
    https://www.bioz.com/result/mg132/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
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    96
    Tocris mg132
    Induction of mesencephalic astrocyte-derived neurotrophic factor expression in the primarily cultured astrocytes. Cells cultured in the DMEM medium containing 5% serum were used as controls ( A to C ). Glial cells were cultured as described in Materials and methods and treated with 1 μg/ml tunicamycin ( D to F ), 10 μM <t>MG132</t> ( G to I ), and serum-free DMEM medium ( J to L ), respectively. Twenty-four hours after treatment, the astrocytes were identified with anti-glial fibrillary acidic protein (anti-GFAP) antibody (red, A , D , G , and J ). Mesencephalic astrocyte-derived neurotrophic factor (MANF) expression was detected with monoclonal anti-MANF antibody (green, B , E , H , and K ). Nuclei were stained with 4 ′ ,6-diamidino-2-phenylindole (blue). The arrows show MANF immune-positive astrocytes after treatment. Scale bar = 50 μm.
    Mg132, supplied by Tocris, used in various techniques. Bioz Stars score: 96/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mg132  (Abcam)
    94
    Abcam mg132
    FBXW7 promotes the ubiquitination of ENO1. ( a ) HEK293T cells were co-transfected with Myc-ubiquitin and wild-type FBXW7, FBXW7ΔF or sh FBXW7 B and then treated with <t>MG132</t> for 6 h. Cell lysates were immunoprecipitated with anti-Myc antibody and
    Mg132, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    LKT Laboratories mg132
    Proteasome subunit up-regulation in β-thalassemia is Nrf1 dependent. (A-B) Wild-type murine fetal liver erythroid cultures were treated for 24 hours with 1μM sulforaphane or 0.1μM <t>MG132</t> to activate Nrf2 or Nrf1, respectively. (A)
    Mg132, supplied by LKT Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Depletion of Smurf2 accelerates degradation of CNKSR2. a Serum starved HEK293 cells were treated with MG132 at the indicated doses and time intervals and expression of CNKSR2 was assessed using western blot indicating proteasome mediated degradation of CNKSR2. b Decline in CNKSR2 expression induced by Smurf2 siRNA was rescued by treating the cells for 4 h with 10 μM MG132 44 h post-transfection. c Depletion of Smurf2 results in enhanced polyubiquitination and proteasomal degradation of CNKSR2. CNKSR2 was immunoprecipitated from MDA-MB-231 cells transfected with Smurf2 siRNA and control siRNA. Cells were treated for 4 h with 10 μM MG132 44 h post-transfection. The corresponding lysates were denatured and immunoprecipitated (IP) with rabbit CNKSR2 antibody, followed by Western blotting (IB) of the immune complexes with a mouse anti-ubiquitin antibody. The input proteins in cell lysates were also probed by the indicated antibodies

    Journal: BMC Cancer

    Article Title: Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression

    doi: 10.1186/s12885-018-4188-x

    Figure Lengend Snippet: Depletion of Smurf2 accelerates degradation of CNKSR2. a Serum starved HEK293 cells were treated with MG132 at the indicated doses and time intervals and expression of CNKSR2 was assessed using western blot indicating proteasome mediated degradation of CNKSR2. b Decline in CNKSR2 expression induced by Smurf2 siRNA was rescued by treating the cells for 4 h with 10 μM MG132 44 h post-transfection. c Depletion of Smurf2 results in enhanced polyubiquitination and proteasomal degradation of CNKSR2. CNKSR2 was immunoprecipitated from MDA-MB-231 cells transfected with Smurf2 siRNA and control siRNA. Cells were treated for 4 h with 10 μM MG132 44 h post-transfection. The corresponding lysates were denatured and immunoprecipitated (IP) with rabbit CNKSR2 antibody, followed by Western blotting (IB) of the immune complexes with a mouse anti-ubiquitin antibody. The input proteins in cell lysates were also probed by the indicated antibodies

    Article Snippet: Lending further support that Smurf2 modulates CNKSR2 expression, we analyzed the role of proteasome mediated degradation in maintaining the stability of CNKSR2 using the proteasome inhibitor, MG132.

    Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation, Multiple Displacement Amplification

    NtERF3, AtERF4, and AtERF8 levels in Arabidopsis plants increased by MG132 and aging. A, GFP florescence of the roots of Pro-35S : NLS-GFP-NtERF3 and MG132. Bars = 50 µm. B and C, Accumulation of NLS-GFP-NtERF3

    Journal: Plant Physiology

    Article Title: A Regulatory Cascade Involving Class II ETHYLENE RESPONSE FACTOR Transcriptional Repressors Operates in the Progression of Leaf Senescence 1A Regulatory Cascade Involving Class II ETHYLENE RESPONSE FACTOR Transcriptional Repressors Operates in the Progression of Leaf Senescence 1 [C]A Regulatory Cascade Involving Class II ETHYLENE RESPONSE FACTOR Transcriptional Repressors Operates in the Progression of Leaf Senescence 1 [C] [W]A Regulatory Cascade Involving Class II ETHYLENE RESPONSE FACTOR Transcriptional Repressors Operates in the Progression of Leaf Senescence 1 [C] [W] [OA]

    doi: 10.1104/pp.113.218115

    Figure Lengend Snippet: NtERF3, AtERF4, and AtERF8 levels in Arabidopsis plants increased by MG132 and aging. A, GFP florescence of the roots of Pro-35S : NLS-GFP-NtERF3 and MG132. Bars = 50 µm. B and C, Accumulation of NLS-GFP-NtERF3

    Article Snippet: For MG 132 treatment, Arabidopsis plants were immersed in a solution containing 50 m m MES (pH 5.7), 0.05% (v/v) Tween 20, and 50 µ m MG132 (Calbiochem) for 6 h in light.

    Techniques:

    FBXL19 interacts with the CDK-Mediator complex in ES cells. ( A ) A representative silver-stained gel for FS2-FBXL19 purification and an empty vector control (EV) purification. Asterisk identifies the band corresponding to FS2-FBXL19 protein. ( B ) In order to visualize FBXL19 protein by western blot in Figure 2—figure supplement 1D and Figure 4B , the Fbxl19 gene was tagged by T7 knock-in in Fbxl19 fl/fl ES cells using the CRISPR Cas9 system. A schematic representation of the generation of the C-terminal T7 knock-in Fbxl19 is shown. HA1/2 indicate the homology arms of the targeting construct. ( C ) Western blot analysis of the expression of T7-FBXL19 from nuclear extract of the generated T7 knock-in ES cell line. ( D ) Western blot analysis of endogenous co-immunoprecipitation (IP) of FBXL19, CDK8 and MED12 from ES cell nuclear extracts. A control IP using a non-specific antibody (α-ΗΑ) was included. ( E ) A schematic illustration of the different FS2-FBXL19 truncation mutants and Western blot analysis of purification of FS2-FBXL19 mutants from HEK293T cells probed with the indicated antibodies. ( F ) Western blot analysis of FS2-FBXL19 and control purifications (EV) probed with the indicated antibodies. ( G ) Western blot analysis of histone extracts generated from Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT) ES cells probed with two different antibodies recognizing ubiquitylated H2B K120 (H2Bub1). H4 was used as a loading control. ( H ) Western blot analysis of nuclear extracts from HEK293T cells transiently transfected with empty vector (EV) or Flag-FBXL19-expressing vector without (-) or following MG132 treatment (+). Blots were probed with the indicated antibodies. TBP was used as loading control.

    Journal: eLife

    Article Title: FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment

    doi: 10.7554/eLife.37084

    Figure Lengend Snippet: FBXL19 interacts with the CDK-Mediator complex in ES cells. ( A ) A representative silver-stained gel for FS2-FBXL19 purification and an empty vector control (EV) purification. Asterisk identifies the band corresponding to FS2-FBXL19 protein. ( B ) In order to visualize FBXL19 protein by western blot in Figure 2—figure supplement 1D and Figure 4B , the Fbxl19 gene was tagged by T7 knock-in in Fbxl19 fl/fl ES cells using the CRISPR Cas9 system. A schematic representation of the generation of the C-terminal T7 knock-in Fbxl19 is shown. HA1/2 indicate the homology arms of the targeting construct. ( C ) Western blot analysis of the expression of T7-FBXL19 from nuclear extract of the generated T7 knock-in ES cell line. ( D ) Western blot analysis of endogenous co-immunoprecipitation (IP) of FBXL19, CDK8 and MED12 from ES cell nuclear extracts. A control IP using a non-specific antibody (α-ΗΑ) was included. ( E ) A schematic illustration of the different FS2-FBXL19 truncation mutants and Western blot analysis of purification of FS2-FBXL19 mutants from HEK293T cells probed with the indicated antibodies. ( F ) Western blot analysis of FS2-FBXL19 and control purifications (EV) probed with the indicated antibodies. ( G ) Western blot analysis of histone extracts generated from Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT) ES cells probed with two different antibodies recognizing ubiquitylated H2B K120 (H2Bub1). H4 was used as a loading control. ( H ) Western blot analysis of nuclear extracts from HEK293T cells transiently transfected with empty vector (EV) or Flag-FBXL19-expressing vector without (-) or following MG132 treatment (+). Blots were probed with the indicated antibodies. TBP was used as loading control.

    Article Snippet: Proteasome inhibition was performed for 4 hr using 10 µM MG132 inhibitor (Sigma-Aldrich).

    Techniques: Staining, Purification, Plasmid Preparation, Western Blot, Knock-In, CRISPR, Construct, Expressing, Generated, Immunoprecipitation, Transfection

    MG132 reversed the high-glucose induced increase of α-SMA. the levels of α-SMA was significantly higher than in CON and were reduced after administration of MG132 and deguelin for the indicted time. α-SMA expression in HMCs was detected by western blotting: HMCs were treated with 5.5 mmol/L (CON) or 30 mmol/L (HG) high glucose for 24 h, 48 h, and 72 h; then, the HG group was treated with MG132 or deguelin. CON: 5.5 mmol/L glucose; HG: 30 mmol/L glucose; MG132: 30 mmol/L glucose with MG132; Deguelin: 30 mmol/L glucose with deguelin; means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: MG132 reversed the high-glucose induced increase of α-SMA. the levels of α-SMA was significantly higher than in CON and were reduced after administration of MG132 and deguelin for the indicted time. α-SMA expression in HMCs was detected by western blotting: HMCs were treated with 5.5 mmol/L (CON) or 30 mmol/L (HG) high glucose for 24 h, 48 h, and 72 h; then, the HG group was treated with MG132 or deguelin. CON: 5.5 mmol/L glucose; HG: 30 mmol/L glucose; MG132: 30 mmol/L glucose with MG132; Deguelin: 30 mmol/L glucose with deguelin; means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques: Expressing, Western Blot

    Effect of MG132 on histology in DN rats. In DN rats, both MG132 and deguelin treatment effectively reduced mesangial cell proliferation, and mesangial matrix accumulation for the indicted time. NC: normal control group at the end of 12 weeks ( A ); DN: diabetic nephropathy rats group at the end of 12 weeks ( B ); MG132: diabetic nephropathy plus MG132 treatment group at the end of 12 weeks ( C ); Deguelin: diabetic nephropathy plus deguelin treatment group at the end of 12 weeks ( D ); Glomerular area (ratio NC group) in all rats ( E ). Representative photomicrographs of neutral formaldehyde (10%)-fixed sections stained with PAS are shown. Magnification ×200. Means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: Effect of MG132 on histology in DN rats. In DN rats, both MG132 and deguelin treatment effectively reduced mesangial cell proliferation, and mesangial matrix accumulation for the indicted time. NC: normal control group at the end of 12 weeks ( A ); DN: diabetic nephropathy rats group at the end of 12 weeks ( B ); MG132: diabetic nephropathy plus MG132 treatment group at the end of 12 weeks ( C ); Deguelin: diabetic nephropathy plus deguelin treatment group at the end of 12 weeks ( D ); Glomerular area (ratio NC group) in all rats ( E ). Representative photomicrographs of neutral formaldehyde (10%)-fixed sections stained with PAS are shown. Magnification ×200. Means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques: Staining

    Effect of MG132 on the NF-κB level in DN rats. In DN rats, the relative mRNA level of NF-κB was significantly higher than in NC rats and was reduced after the administration of MG132 and deguelin for the indicted time ( A ). Likewise, the level of p65 was significantly higher than in NC rats and was reduced after administration of MG132 and deguelin for the indicted time ( B ). NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. Means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: Effect of MG132 on the NF-κB level in DN rats. In DN rats, the relative mRNA level of NF-κB was significantly higher than in NC rats and was reduced after the administration of MG132 and deguelin for the indicted time ( A ). Likewise, the level of p65 was significantly higher than in NC rats and was reduced after administration of MG132 and deguelin for the indicted time ( B ). NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. Means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques:

    Effect of MG132 on inflammatory cytokine expression in DN rats. MCP-1 mRNA expression was examined by RT-PCR ( A ), and MCP-1 ( B ) protein expression levels were determined using Western blot. In DN rats, the levels of MCP-1 were significantly higher than in NC rats and were reduced after administration of MG132 and deguelin for the indicted time. TGF-β1 mRNA expression was examined by RT-PCR ( C ), and TGF-β1 ( D ) protein expression levels were determined using Western blot. In DN rats, the relative levels of TGF-β1 were significantly higher than in NC rats and were reduced after administration of MG132 and deguelin for the indicted time. In DN rats, the concentration of urine MCP-1 was significantly higher than in NC rats and was reduced after administration of MG132 and deguelin for the indicted time ( E ). NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. Means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: Effect of MG132 on inflammatory cytokine expression in DN rats. MCP-1 mRNA expression was examined by RT-PCR ( A ), and MCP-1 ( B ) protein expression levels were determined using Western blot. In DN rats, the levels of MCP-1 were significantly higher than in NC rats and were reduced after administration of MG132 and deguelin for the indicted time. TGF-β1 mRNA expression was examined by RT-PCR ( C ), and TGF-β1 ( D ) protein expression levels were determined using Western blot. In DN rats, the relative levels of TGF-β1 were significantly higher than in NC rats and were reduced after administration of MG132 and deguelin for the indicted time. In DN rats, the concentration of urine MCP-1 was significantly higher than in NC rats and was reduced after administration of MG132 and deguelin for the indicted time ( E ). NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. Means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay

    Effect of MG132 on HMCs proliferation. Exposure to 30 mmol/L glucose activated the proliferation of HMCs, manifesting as an increase of the absorbance value of MTT. Both MG132 and deguelin treatment effectively reduced the proliferation of HMCs. CON: normal glucose medium containing 5.5 mmol/L glucose; HG: high glucose containing 30 mmol/L glucose; MG132: high glucose containing 30 mmol/L glucose with MG132; Deguelin: high glucose containing 30 mmol/L glucose with deguelin. * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: Effect of MG132 on HMCs proliferation. Exposure to 30 mmol/L glucose activated the proliferation of HMCs, manifesting as an increase of the absorbance value of MTT. Both MG132 and deguelin treatment effectively reduced the proliferation of HMCs. CON: normal glucose medium containing 5.5 mmol/L glucose; HG: high glucose containing 30 mmol/L glucose; MG132: high glucose containing 30 mmol/L glucose with MG132; Deguelin: high glucose containing 30 mmol/L glucose with deguelin. * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques: MTT Assay

    Effect of MG132 on sclerotic degree in DN rats. In DN rats, both MG132 and deguelin treatment effectively reduced the sclerotic degree for the indicted time. NC: normal control group at the end of the study (12 weeks); DN: diabetic nephropathy group at the end of the study (12 weeks); MG132: diabetic nephropathy plus MG132 treatment group at the end of the study (12 weeks); Deguelin: diabetic nephropathy plus deguelin treatment group at the end of the study (12 weeks). Means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: Effect of MG132 on sclerotic degree in DN rats. In DN rats, both MG132 and deguelin treatment effectively reduced the sclerotic degree for the indicted time. NC: normal control group at the end of the study (12 weeks); DN: diabetic nephropathy group at the end of the study (12 weeks); MG132: diabetic nephropathy plus MG132 treatment group at the end of the study (12 weeks); Deguelin: diabetic nephropathy plus deguelin treatment group at the end of the study (12 weeks). Means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques:

    Effect of MG132 on the urinary protein excretion rate in DN rats. In DN rats, both MG132 and deguelin treatment effectively reduced urinary protein excretion for the indicted time. NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. Means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: Effect of MG132 on the urinary protein excretion rate in DN rats. In DN rats, both MG132 and deguelin treatment effectively reduced urinary protein excretion for the indicted time. NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. Means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques:

    MG132 reversed the high-glucose induced increase of p-Akt(Ser 473 ). ( A ) p-Akt(Ser 473 ) expression in renal tissue was detected by western blotting: the level of p-Akt(Ser 473 ) in the DN group was significantly higher than in the NC group and was reduced after administration of MG132 and deguelin for the indicted time. NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. ( B ) p-Akt(Ser 473 ) expression in HMCs was detected by western blotting: HMCs was treated with 5.5 mmol/L (CON) or 30 mmol/L (HG) high glucose for 24 h, 48 h, and 72 h; then, the HG group was treated with MG132 or deguelin. CON: 5.5 mmol/L glucose; HG: 30 mmol/L glucose; MG132: 30 mmol/L glucose with MG132; Deguelin: 30 mmol/L glucose with deguelin; means ± SEM; N = 6; * P

    Journal: Scientific Reports

    Article Title: MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy

    doi: 10.1038/s41598-018-38425-2

    Figure Lengend Snippet: MG132 reversed the high-glucose induced increase of p-Akt(Ser 473 ). ( A ) p-Akt(Ser 473 ) expression in renal tissue was detected by western blotting: the level of p-Akt(Ser 473 ) in the DN group was significantly higher than in the NC group and was reduced after administration of MG132 and deguelin for the indicted time. NC: normal control group; DN: diabetic nephropathy group; MG132: diabetic nephropathy plus MG132 treatment group; Deguelin: diabetic nephropathy plus deguelin treatment group. ( B ) p-Akt(Ser 473 ) expression in HMCs was detected by western blotting: HMCs was treated with 5.5 mmol/L (CON) or 30 mmol/L (HG) high glucose for 24 h, 48 h, and 72 h; then, the HG group was treated with MG132 or deguelin. CON: 5.5 mmol/L glucose; HG: 30 mmol/L glucose; MG132: 30 mmol/L glucose with MG132; Deguelin: 30 mmol/L glucose with deguelin; means ± SEM; N = 6; * P

    Article Snippet: All DN rats (n = 54) were randomly divided into three subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.) either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established (week 0).

    Techniques: Expressing, Western Blot

    MDC staining in root cells. Seedlings treated with 0 (a), 20 µM (b), 40 µM (c), or 80 µM (d) MG132 were stained with MDC (Blue) and FM 4–64 (Red) and examined with a Zeiss confocal. The data show the accumulation of MDC in root cells in response to MG132 treatment. Bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: MDC staining in root cells. Seedlings treated with 0 (a), 20 µM (b), 40 µM (c), or 80 µM (d) MG132 were stained with MDC (Blue) and FM 4–64 (Red) and examined with a Zeiss confocal. The data show the accumulation of MDC in root cells in response to MG132 treatment. Bar = 20 µm.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques: Staining

    Effects of MG132 on root-meristem size. Roots treated with 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d) were stained with FM 4–64, and longitudinal views were obtained using a Zeiss confocal. The results show reduced root-meristem size (indicated by arrows) and accelerated cell differentiation (indicated by stars) in response to MG132 treatment. Bars = 20 µm.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: Effects of MG132 on root-meristem size. Roots treated with 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d) were stained with FM 4–64, and longitudinal views were obtained using a Zeiss confocal. The results show reduced root-meristem size (indicated by arrows) and accelerated cell differentiation (indicated by stars) in response to MG132 treatment. Bars = 20 µm.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques: Staining, Cell Differentiation

    MG132 alters cell length in the maturation zone. Roots treated with 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d) were stained with FM 4–64, and longitudinal views were obtained using a Zeiss confocal. The results show that MG132 alters cell length in the maturation zone (indicated by arrows). Bars = 20 µm.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: MG132 alters cell length in the maturation zone. Roots treated with 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d) were stained with FM 4–64, and longitudinal views were obtained using a Zeiss confocal. The results show that MG132 alters cell length in the maturation zone (indicated by arrows). Bars = 20 µm.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques: Staining

    GFP-fusion cyclin B analysis. Arabidopsis carrying GFP-fusion cyclin B were treated with 0 (a), 20 µM (b), 40 µM (c), or 80 µM (d) MG132, respectively. Longitudinal views of the GFP (left) and FM 4–64 (middle) fluorescent emissions were collected separately and then merged (right), using a Zeiss confocal. Figures show accumulation of cyclin B in root-meristemic zone in response to MG132 treatment. Bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: GFP-fusion cyclin B analysis. Arabidopsis carrying GFP-fusion cyclin B were treated with 0 (a), 20 µM (b), 40 µM (c), or 80 µM (d) MG132, respectively. Longitudinal views of the GFP (left) and FM 4–64 (middle) fluorescent emissions were collected separately and then merged (right), using a Zeiss confocal. Figures show accumulation of cyclin B in root-meristemic zone in response to MG132 treatment. Bar = 20 µm.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques:

    Effects of different inhibitors on primary root elongation. a–d. Cold-pretreated seeds were cultured in 1/2 MS with 1% Suc for 24 h, and then uniform seedlings of similar size and primary root length were transferred to the same fresh medium containing 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d), respectively, for additional 48 h. Bars = 1000 µm. e–h. Cold-pretreated seeds were cultured in 1/2 MS with 1% Suc for 24 h, and then uniform seedlings of similar size and primary root length were transferred to the same fresh medium containing various concentrations of MG 132 (e), MG115 (f), E-64 (g) and DMSO (h), for additional 48 days. The values are the means ± SD of about 15 seedlings of each treatment. The experiment was repeated thrice with consistent results.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: Effects of different inhibitors on primary root elongation. a–d. Cold-pretreated seeds were cultured in 1/2 MS with 1% Suc for 24 h, and then uniform seedlings of similar size and primary root length were transferred to the same fresh medium containing 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d), respectively, for additional 48 h. Bars = 1000 µm. e–h. Cold-pretreated seeds were cultured in 1/2 MS with 1% Suc for 24 h, and then uniform seedlings of similar size and primary root length were transferred to the same fresh medium containing various concentrations of MG 132 (e), MG115 (f), E-64 (g) and DMSO (h), for additional 48 days. The values are the means ± SD of about 15 seedlings of each treatment. The experiment was repeated thrice with consistent results.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques: Cell Culture, Mass Spectrometry

    LysoTracker staining in root cells. Seedlings treated with 0 (a), 20 µM (b), 40 µM (c), or 80 µM (d) MG132 were stained with LysoTracker (Green) and FM 4–64 (Red), and examined with a Zeiss confocal. The data show the accumulation of LysoTracker in root cells in response to MG132 treatment. Bar = 50 µm.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: LysoTracker staining in root cells. Seedlings treated with 0 (a), 20 µM (b), 40 µM (c), or 80 µM (d) MG132 were stained with LysoTracker (Green) and FM 4–64 (Red), and examined with a Zeiss confocal. The data show the accumulation of LysoTracker in root cells in response to MG132 treatment. Bar = 50 µm.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques: Staining

    Immunofluorescence labeling of ubiquitinated proteins in root cells. Semi-thin sections of roots treated with 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d) were incubated with anti-ubiquitin antibody and FITC-IgG, and examined with a Zeiss confocal. Arrows show the MG132-induced accumulation of ubiquitinated proteins in vacuoles. Bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    doi: 10.1371/journal.pone.0045673

    Figure Lengend Snippet: Immunofluorescence labeling of ubiquitinated proteins in root cells. Semi-thin sections of roots treated with 0 (a), 20 µM MG132 (b), 40 µM MG132 (c) or 80 µM MG132 (d) were incubated with anti-ubiquitin antibody and FITC-IgG, and examined with a Zeiss confocal. Arrows show the MG132-induced accumulation of ubiquitinated proteins in vacuoles. Bar = 20 µm.

    Article Snippet: Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma.

    Techniques: Immunofluorescence, Labeling, Incubation

    Chromosome alignment configuration in fresh oocytes and cryopreserved oocytes from untreated and 10 mM caffeine +10 μM MG132 (CA+MG) groups (A) Comparison of chromosome alignment configurations. Anti-tubulin immunostaining (red) indicates spindles and DAPI staining (blue) indicates chromosomes. Yellow arrow indicates an abnormal chromosome alignment in the untreated group. Scale bar = 10 μm. (B) Percentage of oocytes derived from vitrified/warmed oocytes (* P

    Journal: Molecules and Cells

    Article Title: Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer

    doi: 10.14348/molcells.2017.0184

    Figure Lengend Snippet: Chromosome alignment configuration in fresh oocytes and cryopreserved oocytes from untreated and 10 mM caffeine +10 μM MG132 (CA+MG) groups (A) Comparison of chromosome alignment configurations. Anti-tubulin immunostaining (red) indicates spindles and DAPI staining (blue) indicates chromosomes. Yellow arrow indicates an abnormal chromosome alignment in the untreated group. Scale bar = 10 μm. (B) Percentage of oocytes derived from vitrified/warmed oocytes (* P

    Article Snippet: MII oocytes were collected and vitrified under four experimental conditions: untreated (control), 10 mM caffeine-supplemented (CA, Sigma-Aldrich), 10 μM MG132-supplemented (MG, Calbiochem), and 10 mM caffeine +10 μM MG132-supplemented (CA+MG).

    Techniques: Immunostaining, Staining, Derivative Assay

    Effects of caffeine/MG132 on MPF and MAPK activity in vitrified/warmed mouse oocytes Vitrified/warmed oocytes were pre-incubated in CPA solution without supplements (untreated control) or containing 10 mM caffeine (CA), 10 μM MG132 (MG) and 10 mM caffeine + 10 μM MG132 (CA+MG) for 2 h, and then collected in 5 μl of sample buffer before vitrification/warming to assess kinase activity. (A) Results of enzyme-linked immunosorbent assays for Cdc2 kinase. (B) Changes in MPF activity in fresh oocytes and oocytes in cryopreserved untreated control, CA, MG and CA+MG groups. (C–D) ERK and pERK expression were not significantly different among groups. Western blots showed similar changes in expression. Thirty oocytes were analyzed for each group (n = 5 replicates/group). Data are shown as the means ± SEM.

    Journal: Molecules and Cells

    Article Title: Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer

    doi: 10.14348/molcells.2017.0184

    Figure Lengend Snippet: Effects of caffeine/MG132 on MPF and MAPK activity in vitrified/warmed mouse oocytes Vitrified/warmed oocytes were pre-incubated in CPA solution without supplements (untreated control) or containing 10 mM caffeine (CA), 10 μM MG132 (MG) and 10 mM caffeine + 10 μM MG132 (CA+MG) for 2 h, and then collected in 5 μl of sample buffer before vitrification/warming to assess kinase activity. (A) Results of enzyme-linked immunosorbent assays for Cdc2 kinase. (B) Changes in MPF activity in fresh oocytes and oocytes in cryopreserved untreated control, CA, MG and CA+MG groups. (C–D) ERK and pERK expression were not significantly different among groups. Western blots showed similar changes in expression. Thirty oocytes were analyzed for each group (n = 5 replicates/group). Data are shown as the means ± SEM.

    Article Snippet: MII oocytes were collected and vitrified under four experimental conditions: untreated (control), 10 mM caffeine-supplemented (CA, Sigma-Aldrich), 10 μM MG132-supplemented (MG, Calbiochem), and 10 mM caffeine +10 μM MG132-supplemented (CA+MG).

    Techniques: Activity Assay, Incubation, Expressing, Western Blot

    Comparison of implantation rates in untreated, 10 mM caffeine (CA) and 10 mM caffeine + 10 μM MG132 (CA+MG) treatment groups (A, B) Implantation sac rate of in vitro -fertilized 2-cell embryos derived from vitrified/warmed oocytes (untreated group: left uterus; CA group: right uterus). (C, D) Implantation sac rate of in vitro -fertilized 2-cell embryos derived from vitrified/warmed oocytes (untreated group: left uterus; CA + MG treatment group: right uterus). (* P

    Journal: Molecules and Cells

    Article Title: Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer

    doi: 10.14348/molcells.2017.0184

    Figure Lengend Snippet: Comparison of implantation rates in untreated, 10 mM caffeine (CA) and 10 mM caffeine + 10 μM MG132 (CA+MG) treatment groups (A, B) Implantation sac rate of in vitro -fertilized 2-cell embryos derived from vitrified/warmed oocytes (untreated group: left uterus; CA group: right uterus). (C, D) Implantation sac rate of in vitro -fertilized 2-cell embryos derived from vitrified/warmed oocytes (untreated group: left uterus; CA + MG treatment group: right uterus). (* P

    Article Snippet: MII oocytes were collected and vitrified under four experimental conditions: untreated (control), 10 mM caffeine-supplemented (CA, Sigma-Aldrich), 10 μM MG132-supplemented (MG, Calbiochem), and 10 mM caffeine +10 μM MG132-supplemented (CA+MG).

    Techniques: In Vitro, Derivative Assay

    Comparison of number of total cells, ICM cells, and TE cells (A) Total cells, ICM cells, and TE cells were counted in fresh oocytes and cryopreserved oocytes from untreated, 10 mM caffeine (CA), 10 μM MG132 (MG), and 10 mM, caffeine +10 μM MG132 (CA+MG) groups. Number of total cells, ICM cells, and TE cells in blastocysts derived from vitrified/warmed oocytes. Blastocysts were observed 120 h after IVF. ICM cells were stained with anti-Oct3/4(green), and total cells were stained nucleus (DAPI, blue) and actin-phalloidin (red). (B) Means of total cell numbers, inner cell mass (ICM), and trophectoderm (TE) of blastocysts from fresh oocytes and cryopreserved oocytes. Differences were not statistically significant. Data are shown as the means ± SEM.

    Journal: Molecules and Cells

    Article Title: Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer

    doi: 10.14348/molcells.2017.0184

    Figure Lengend Snippet: Comparison of number of total cells, ICM cells, and TE cells (A) Total cells, ICM cells, and TE cells were counted in fresh oocytes and cryopreserved oocytes from untreated, 10 mM caffeine (CA), 10 μM MG132 (MG), and 10 mM, caffeine +10 μM MG132 (CA+MG) groups. Number of total cells, ICM cells, and TE cells in blastocysts derived from vitrified/warmed oocytes. Blastocysts were observed 120 h after IVF. ICM cells were stained with anti-Oct3/4(green), and total cells were stained nucleus (DAPI, blue) and actin-phalloidin (red). (B) Means of total cell numbers, inner cell mass (ICM), and trophectoderm (TE) of blastocysts from fresh oocytes and cryopreserved oocytes. Differences were not statistically significant. Data are shown as the means ± SEM.

    Article Snippet: MII oocytes were collected and vitrified under four experimental conditions: untreated (control), 10 mM caffeine-supplemented (CA, Sigma-Aldrich), 10 μM MG132-supplemented (MG, Calbiochem), and 10 mM caffeine +10 μM MG132-supplemented (CA+MG).

    Techniques: Derivative Assay, Staining

    THOC7 is involved in the (mitochondrial antiviral signaling (MAVS) signalosome and drives proteasomal degradation of TBK1. ( A ) THOC7 was involved in forming the MAVS signaling complex. 293T cells were seeded into 100-mm dishes and transfected with Flag-tagged THOC7 or empty control plasmids (10 μg) and HA-tagged RLR signaling molecules (10 μg) as indicated. After 12 h of transfection, the cells were treated with or without SeV for 12 h. Co-immunoprecipitation and immunoblot analysis was performed with the indicated antibodies. ( B ) THOC7 promoted degradation of exogenous and endogenous TBK1 in a dose-dependent manner. 293T cells were seeded into 6-well plates and transfected with increasing amounts of Flag-THOC7 (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg), and HA-TBK1 (2 μg). Twenty-four hours after transfection, immunoblot analysis was performed with the indicated antibodies. ( C ) MG132 restored the TBK1 protein level in the presence of THOC7. 293T cells were seeded into 6-well plates and transfected with Flag-THOC7 (2 μg) and HA-TBK1 (1.5 μg). MG132 and CHX were added after 6 h of transfection and the cells were harvested for analysis after 24 h. ( D , E ) THOC7 increased the K48-linked ubiquitination of TBK1. 293T cells were seeded into 100-mm dishes and transfected with the indicated plasmids (8 μg each). Similar co-immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Quantification of western blotting bands ( A , C , E , F ) from three independent experiments was performed with Image J software. Error bars indicate SD. n = 3. *, p

    Journal: Viruses

    Article Title: THO Complex Subunit 7 Homolog Negatively Regulates Cellular Antiviral Response against RNA Viruses by Targeting TBK1

    doi: 10.3390/v11020158

    Figure Lengend Snippet: THOC7 is involved in the (mitochondrial antiviral signaling (MAVS) signalosome and drives proteasomal degradation of TBK1. ( A ) THOC7 was involved in forming the MAVS signaling complex. 293T cells were seeded into 100-mm dishes and transfected with Flag-tagged THOC7 or empty control plasmids (10 μg) and HA-tagged RLR signaling molecules (10 μg) as indicated. After 12 h of transfection, the cells were treated with or without SeV for 12 h. Co-immunoprecipitation and immunoblot analysis was performed with the indicated antibodies. ( B ) THOC7 promoted degradation of exogenous and endogenous TBK1 in a dose-dependent manner. 293T cells were seeded into 6-well plates and transfected with increasing amounts of Flag-THOC7 (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg), and HA-TBK1 (2 μg). Twenty-four hours after transfection, immunoblot analysis was performed with the indicated antibodies. ( C ) MG132 restored the TBK1 protein level in the presence of THOC7. 293T cells were seeded into 6-well plates and transfected with Flag-THOC7 (2 μg) and HA-TBK1 (1.5 μg). MG132 and CHX were added after 6 h of transfection and the cells were harvested for analysis after 24 h. ( D , E ) THOC7 increased the K48-linked ubiquitination of TBK1. 293T cells were seeded into 100-mm dishes and transfected with the indicated plasmids (8 μg each). Similar co-immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Quantification of western blotting bands ( A , C , E , F ) from three independent experiments was performed with Image J software. Error bars indicate SD. n = 3. *, p

    Article Snippet: The results showed that MG132 efficiently restored the TBK1 protein level in the presence of THOC7 ( C).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Software

    HPIV3 C protein promotes NLRP3 degradation via the proteasome. (A) Cell lysates collected from THP-1 cells expressing FLAG-C were subjected to Western blotting with anti-NLRP3 antibody to examine the status of endogenous NLRP3 protein expression. The blot was also probed with anti-FLAG antibody to detect the expression of the FLAG-C protein. (B) Cell lysates collected from THP-1 cells expressing FLAG-HPIV1 C were subjected to Western blotting with anti-NLRP3 antibody to examine the status of endogenous NLRP3 protein expression. The blot was also probed with anti-FLAG antibody to detect the expression of the FLAG-HPIV1 C protein. (C) Cell lysates collected from HEK293 cells coexpressing Myc-NLRP3 and FLAG-C in the presence of the vehicle (DMSO) or the proteasome inhibitor MG132 (10 μM) were subjected to Western blotting with anti-Myc antibody. In panels A to C, protein bands corresponding to NLRP3 and actin were quantified to calculate the NLRP3/actin ratio (relative intensity). Immunoblots (A and C) are representative of data from two independent experiments with similar results. FLAG, empty FLAG.

    Journal: Journal of Virology

    Article Title: Inflammasome Antagonism by Human Parainfluenza Virus Type 3 C Protein

    doi: 10.1128/JVI.01776-17

    Figure Lengend Snippet: HPIV3 C protein promotes NLRP3 degradation via the proteasome. (A) Cell lysates collected from THP-1 cells expressing FLAG-C were subjected to Western blotting with anti-NLRP3 antibody to examine the status of endogenous NLRP3 protein expression. The blot was also probed with anti-FLAG antibody to detect the expression of the FLAG-C protein. (B) Cell lysates collected from THP-1 cells expressing FLAG-HPIV1 C were subjected to Western blotting with anti-NLRP3 antibody to examine the status of endogenous NLRP3 protein expression. The blot was also probed with anti-FLAG antibody to detect the expression of the FLAG-HPIV1 C protein. (C) Cell lysates collected from HEK293 cells coexpressing Myc-NLRP3 and FLAG-C in the presence of the vehicle (DMSO) or the proteasome inhibitor MG132 (10 μM) were subjected to Western blotting with anti-Myc antibody. In panels A to C, protein bands corresponding to NLRP3 and actin were quantified to calculate the NLRP3/actin ratio (relative intensity). Immunoblots (A and C) are representative of data from two independent experiments with similar results. FLAG, empty FLAG.

    Article Snippet: In several experiments, transfected cells were also treated with the proteasome inhibitor MG132 (10 to 50 μM) (InvivoGen).

    Techniques: Expressing, Western Blot

    ERK interacts with FBW7. (A , B) Immunoblot (IB) analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) from 293T cells transfected with Flag-WT-FBW7, or HA-ERK1 or together. Thirty hours post-transfection, cells were pretreated with 15 μM MG132 for 10 h before harvesting. (C , D) Immunoblot analysis of WCL and immunoprecipitates from the indicated SW1990 cells stably expressing the Flag-ERK1 construct. Cells were pretreated with 15 μM MG132 for 10 h before harvesting. (E) SW1990 cell extracts were immunoprecipitated with an antibody against FBW7 or control IgG and analyzed by immunoblot analysis.

    Journal: Cell Research

    Article Title: ERK kinase phosphorylates and destabilizes the tumor suppressor FBW7 in pancreatic cancer

    doi: 10.1038/cr.2015.30

    Figure Lengend Snippet: ERK interacts with FBW7. (A , B) Immunoblot (IB) analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) from 293T cells transfected with Flag-WT-FBW7, or HA-ERK1 or together. Thirty hours post-transfection, cells were pretreated with 15 μM MG132 for 10 h before harvesting. (C , D) Immunoblot analysis of WCL and immunoprecipitates from the indicated SW1990 cells stably expressing the Flag-ERK1 construct. Cells were pretreated with 15 μM MG132 for 10 h before harvesting. (E) SW1990 cell extracts were immunoprecipitated with an antibody against FBW7 or control IgG and analyzed by immunoblot analysis.

    Article Snippet: UO126, AZD6244, trametinib, SCH772984 and MG132 were purchased from Selleck.

    Techniques: Transfection, Stable Transfection, Expressing, Construct, Immunoprecipitation

    ERK phosphorylates FBW7 at T205. (A) Detection of in vivo FBW7 T205 phosphorylation by mass spectrometry analysis. (B) PANC-1 cells were pretreated with the proteasome inhibitor MG132 and trametinib, as indicated, overnight before harvest. Endogenous FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitates (IP). (C) 293T cells were transfected with Flag-WT-FBW7. Thirty hours posttransfection, cells were pretreated with MG132 and various MEK/ERK inhibitors overnight before harvesting. FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitation. (D - F) 293T cells were transfected with Flag-WT-FBW7 and Flag-T205A-FBW7, together with HA-MEK1 and HA-ERK1 constructs, respectively. Wild-type (WT) and the constitutively active (CA) form of MEK1 were used as indicated. Thirty hours post-transfection, cells were pretreated with MG132 overnight before harvesting. FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitation. Immunoprecipitates were treated with CIP for 20 min at room temperature, and the reaction was stopped by the addition of SDS loading buffer (F) . (G) Recombinant GST-WT-FBW7 and GST-T205A-FBW7 proteins were incubated with HA-ERK1 kinase in the presence of ATP and the kinase reaction buffer. Thirty minutes later, the reaction was stopped by the addition of the loading buffer. The kinase reaction products were resolved by SDS-PAGE, and FBW7 phosphorylation was detected by the specific p-FBW7 antibody.

    Journal: Cell Research

    Article Title: ERK kinase phosphorylates and destabilizes the tumor suppressor FBW7 in pancreatic cancer

    doi: 10.1038/cr.2015.30

    Figure Lengend Snippet: ERK phosphorylates FBW7 at T205. (A) Detection of in vivo FBW7 T205 phosphorylation by mass spectrometry analysis. (B) PANC-1 cells were pretreated with the proteasome inhibitor MG132 and trametinib, as indicated, overnight before harvest. Endogenous FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitates (IP). (C) 293T cells were transfected with Flag-WT-FBW7. Thirty hours posttransfection, cells were pretreated with MG132 and various MEK/ERK inhibitors overnight before harvesting. FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitation. (D - F) 293T cells were transfected with Flag-WT-FBW7 and Flag-T205A-FBW7, together with HA-MEK1 and HA-ERK1 constructs, respectively. Wild-type (WT) and the constitutively active (CA) form of MEK1 were used as indicated. Thirty hours post-transfection, cells were pretreated with MG132 overnight before harvesting. FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitation. Immunoprecipitates were treated with CIP for 20 min at room temperature, and the reaction was stopped by the addition of SDS loading buffer (F) . (G) Recombinant GST-WT-FBW7 and GST-T205A-FBW7 proteins were incubated with HA-ERK1 kinase in the presence of ATP and the kinase reaction buffer. Thirty minutes later, the reaction was stopped by the addition of the loading buffer. The kinase reaction products were resolved by SDS-PAGE, and FBW7 phosphorylation was detected by the specific p-FBW7 antibody.

    Article Snippet: UO126, AZD6244, trametinib, SCH772984 and MG132 were purchased from Selleck.

    Techniques: In Vivo, Mass Spectrometry, Transfection, Immunoprecipitation, Construct, Recombinant, Incubation, SDS Page

    ERK destabilizes FBW7 by promoting its ubiquitination. (A) Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates of 293T cells transfected with HA-ERK1 together with Myc-Ub and various Flag-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (B) Immunoblot (IB) analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with wild-type HA-ERK1 or kinase-dead (KD) mutant together with Myc-Ub and Flag-WT-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (C) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of Hela cells transfected with Flag-WT-FBW7 together with Myc-Ub and siRNA against ERK1/2. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (D) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with Flag-WT-FBW7 and Myc-Ub constructs. Thirty hours posttransfection, cells were treated with MG132 and trametinib (where indicated) overnight before harvest. (E) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with HA-KRAS G12D together with Myc-Ub and Flag-WT-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (F) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with Flag-WT-FBW7 and HA-ERK1 together with Myc-Ub and siRNA oligos against Pin1. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest.

    Journal: Cell Research

    Article Title: ERK kinase phosphorylates and destabilizes the tumor suppressor FBW7 in pancreatic cancer

    doi: 10.1038/cr.2015.30

    Figure Lengend Snippet: ERK destabilizes FBW7 by promoting its ubiquitination. (A) Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates of 293T cells transfected with HA-ERK1 together with Myc-Ub and various Flag-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (B) Immunoblot (IB) analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with wild-type HA-ERK1 or kinase-dead (KD) mutant together with Myc-Ub and Flag-WT-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (C) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of Hela cells transfected with Flag-WT-FBW7 together with Myc-Ub and siRNA against ERK1/2. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (D) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with Flag-WT-FBW7 and Myc-Ub constructs. Thirty hours posttransfection, cells were treated with MG132 and trametinib (where indicated) overnight before harvest. (E) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with HA-KRAS G12D together with Myc-Ub and Flag-WT-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (F) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with Flag-WT-FBW7 and HA-ERK1 together with Myc-Ub and siRNA oligos against Pin1. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest.

    Article Snippet: UO126, AZD6244, trametinib, SCH772984 and MG132 were purchased from Selleck.

    Techniques: Transfection, Construct, Mutagenesis

    Ubiquitin occupancy, non-ubiquitin occupied and total protein ratios were generated for all partially ubiquitinated peptides detected in MG132 and DMSO control treated samples. a Relative ubiquitinated, non-ubiquitinated and protein ratios for all partially ubiquitinated peptides in the MG132 treated vs. native state. b Percent ubiquitin occupancy for partially ubiquitinated peptides in the MG132 treated and native conditions. c Relative ubiquitinated, non-ubiquitinated and protein ratios for all partially ubiquitinated peptides in the DMSO treated vs. native state. d Percent ubiquitin occupancy for partially ubiquitinated peptides in the DMSO treated and native conditions

    Journal: Clinical Proteomics

    Article Title: Proteomic analysis of degradation ubiquitin signaling by ubiquitin occupancy changes responding to 26S proteasome inhibition

    doi: 10.1186/s12014-020-9265-x

    Figure Lengend Snippet: Ubiquitin occupancy, non-ubiquitin occupied and total protein ratios were generated for all partially ubiquitinated peptides detected in MG132 and DMSO control treated samples. a Relative ubiquitinated, non-ubiquitinated and protein ratios for all partially ubiquitinated peptides in the MG132 treated vs. native state. b Percent ubiquitin occupancy for partially ubiquitinated peptides in the MG132 treated and native conditions. c Relative ubiquitinated, non-ubiquitinated and protein ratios for all partially ubiquitinated peptides in the DMSO treated vs. native state. d Percent ubiquitin occupancy for partially ubiquitinated peptides in the DMSO treated and native conditions

    Article Snippet: The MG132 to native ubiquitinated, non-ubiquitinated and protein ratios are calculated for each peptide using the ubiquitin enriched and global data sets (Figs. b, a, c).

    Techniques: Generated

    Overview of ubiquitinated proteins identified in SKOV3 ovarian cancer cells with MG132 and DMSO control treatment. a Cellular distribution of ubiquitinated proteins identified in MG132 treated SKOV3 cells. b Cellular distribution of ubiquitinated peptides observed in DMSO control treated SKOV3 cells. c Functional classification of ubiquitinated proteins detected in MG132 treated SKOV3 cells. d Functional classification of ubiquitinated proteins detected in DMSO control treated SKOV3 cells

    Journal: Clinical Proteomics

    Article Title: Proteomic analysis of degradation ubiquitin signaling by ubiquitin occupancy changes responding to 26S proteasome inhibition

    doi: 10.1186/s12014-020-9265-x

    Figure Lengend Snippet: Overview of ubiquitinated proteins identified in SKOV3 ovarian cancer cells with MG132 and DMSO control treatment. a Cellular distribution of ubiquitinated proteins identified in MG132 treated SKOV3 cells. b Cellular distribution of ubiquitinated peptides observed in DMSO control treated SKOV3 cells. c Functional classification of ubiquitinated proteins detected in MG132 treated SKOV3 cells. d Functional classification of ubiquitinated proteins detected in DMSO control treated SKOV3 cells

    Article Snippet: The MG132 to native ubiquitinated, non-ubiquitinated and protein ratios are calculated for each peptide using the ubiquitin enriched and global data sets (Figs. b, a, c).

    Techniques: Functional Assay

    Experimental approach and computational analysis for the assessment of ubiquitin occupancy and total protein ratios. a Experimental approach: SILAC LC–MS/MS was used to identify changes in the ubiquitinome of SKOV3 ovarian cancer cells in response to 26S proteasome inhibition by MG132. Cells were cultured in either light or heavy (containing isotopically labeled arginine and lysine residues) RPMI 1640 media. Light cells were treated with either DMSO negative control of MG132 26S proteasome inhibitor, while cells grown in heavy media remained in a native, untreated state. Light and heavy lysates were combined in a 1:1 ratio and after trypsin digestion were either fractionated by bRPLC or ubiquitin-enriched and then fractionated, corresponding to the global and ubiquitinome data sets respectively. Peptides in the global and ubiquitin-enriched samples were detected by LC–MS/MS analysis, which successfully distinguished peptides as originating from the treated (light) or native (heavy) samples based on their m/z ratio. b Partially ubiquitinated peptides can exist in one of two forms, ubiquitin occupied or non-ubiquitinated and the percent abundance of both has to equal 100%. Relative ubiquitinated, non-ubiquitinated and protein ratios (Rub, Rnon-ub and Rprotein) were calculated for all partially ubiquitinated peptides in the MG132 treated (State 2) vs. native (State 1) condition. These rations were subsequently used to determine percent ubiquitin occupancy in State 1 (Pub1), which was then utilized for calculating percent ubiquitin occupancy in State 1 (Pub2)

    Journal: Clinical Proteomics

    Article Title: Proteomic analysis of degradation ubiquitin signaling by ubiquitin occupancy changes responding to 26S proteasome inhibition

    doi: 10.1186/s12014-020-9265-x

    Figure Lengend Snippet: Experimental approach and computational analysis for the assessment of ubiquitin occupancy and total protein ratios. a Experimental approach: SILAC LC–MS/MS was used to identify changes in the ubiquitinome of SKOV3 ovarian cancer cells in response to 26S proteasome inhibition by MG132. Cells were cultured in either light or heavy (containing isotopically labeled arginine and lysine residues) RPMI 1640 media. Light cells were treated with either DMSO negative control of MG132 26S proteasome inhibitor, while cells grown in heavy media remained in a native, untreated state. Light and heavy lysates were combined in a 1:1 ratio and after trypsin digestion were either fractionated by bRPLC or ubiquitin-enriched and then fractionated, corresponding to the global and ubiquitinome data sets respectively. Peptides in the global and ubiquitin-enriched samples were detected by LC–MS/MS analysis, which successfully distinguished peptides as originating from the treated (light) or native (heavy) samples based on their m/z ratio. b Partially ubiquitinated peptides can exist in one of two forms, ubiquitin occupied or non-ubiquitinated and the percent abundance of both has to equal 100%. Relative ubiquitinated, non-ubiquitinated and protein ratios (Rub, Rnon-ub and Rprotein) were calculated for all partially ubiquitinated peptides in the MG132 treated (State 2) vs. native (State 1) condition. These rations were subsequently used to determine percent ubiquitin occupancy in State 1 (Pub1), which was then utilized for calculating percent ubiquitin occupancy in State 1 (Pub2)

    Article Snippet: The MG132 to native ubiquitinated, non-ubiquitinated and protein ratios are calculated for each peptide using the ubiquitin enriched and global data sets (Figs. b, a, c).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Inhibition, Cell Culture, Labeling, Negative Control

    Induction of mesencephalic astrocyte-derived neurotrophic factor expression in the primarily cultured astrocytes. Cells cultured in the DMEM medium containing 5% serum were used as controls ( A to C ). Glial cells were cultured as described in Materials and methods and treated with 1 μg/ml tunicamycin ( D to F ), 10 μM MG132 ( G to I ), and serum-free DMEM medium ( J to L ), respectively. Twenty-four hours after treatment, the astrocytes were identified with anti-glial fibrillary acidic protein (anti-GFAP) antibody (red, A , D , G , and J ). Mesencephalic astrocyte-derived neurotrophic factor (MANF) expression was detected with monoclonal anti-MANF antibody (green, B , E , H , and K ). Nuclei were stained with 4 ′ ,6-diamidino-2-phenylindole (blue). The arrows show MANF immune-positive astrocytes after treatment. Scale bar = 50 μm.

    Journal: Journal of Neuroinflammation

    Article Title: Upregulation of mesencephalic astrocyte-derived neurotrophic factor in glial cells is associated with ischemia-induced glial activation

    doi: 10.1186/1742-2094-9-254

    Figure Lengend Snippet: Induction of mesencephalic astrocyte-derived neurotrophic factor expression in the primarily cultured astrocytes. Cells cultured in the DMEM medium containing 5% serum were used as controls ( A to C ). Glial cells were cultured as described in Materials and methods and treated with 1 μg/ml tunicamycin ( D to F ), 10 μM MG132 ( G to I ), and serum-free DMEM medium ( J to L ), respectively. Twenty-four hours after treatment, the astrocytes were identified with anti-glial fibrillary acidic protein (anti-GFAP) antibody (red, A , D , G , and J ). Mesencephalic astrocyte-derived neurotrophic factor (MANF) expression was detected with monoclonal anti-MANF antibody (green, B , E , H , and K ). Nuclei were stained with 4 ′ ,6-diamidino-2-phenylindole (blue). The arrows show MANF immune-positive astrocytes after treatment. Scale bar = 50 μm.

    Article Snippet: The number of dead cells increased after treatment with tunicamycin (Figure ) or MG132 (Figure ), or under serum-free culture condition (Figure ), suggesting that ER stress induces glial cell death.

    Techniques: Derivative Assay, Expressing, Cell Culture, Staining

    Endoplasmic reticulum stress and mesencephalic astrocyte-derived neurotrophic factor expression in cultured primary glial cells. ( A to D ) Glial cells in the mixed culture were treated as indicated (serum+ control, DMEM medium containing 5% serum; Tm, tunicamycin, 1 μg/ml; MG132, 10 μM; serum–, serum-free DMEM medium). Twenty-four hours after treatment, the cells were collected and processed for RT-PCR and immunoblotting (IB). Levels of mRNAs ( A ) and proteins ( C ) were quantitated and normalized by actin. The quantitative data in ( A ) and ( C ) are shown in ( B ) and ( D ), respectively. Values expressed as mean ± standard error of the mean of three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: Upregulation of mesencephalic astrocyte-derived neurotrophic factor in glial cells is associated with ischemia-induced glial activation

    doi: 10.1186/1742-2094-9-254

    Figure Lengend Snippet: Endoplasmic reticulum stress and mesencephalic astrocyte-derived neurotrophic factor expression in cultured primary glial cells. ( A to D ) Glial cells in the mixed culture were treated as indicated (serum+ control, DMEM medium containing 5% serum; Tm, tunicamycin, 1 μg/ml; MG132, 10 μM; serum–, serum-free DMEM medium). Twenty-four hours after treatment, the cells were collected and processed for RT-PCR and immunoblotting (IB). Levels of mRNAs ( A ) and proteins ( C ) were quantitated and normalized by actin. The quantitative data in ( A ) and ( C ) are shown in ( B ) and ( D ), respectively. Values expressed as mean ± standard error of the mean of three independent experiments. * P

    Article Snippet: The number of dead cells increased after treatment with tunicamycin (Figure ) or MG132 (Figure ), or under serum-free culture condition (Figure ), suggesting that ER stress induces glial cell death.

    Techniques: Derivative Assay, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    FBXW7 promotes the ubiquitination of ENO1. ( a ) HEK293T cells were co-transfected with Myc-ubiquitin and wild-type FBXW7, FBXW7ΔF or sh FBXW7 B and then treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-Myc antibody and

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: FBXW7 negatively regulates ENO1 expression and function in colorectal cancer

    doi: 10.1038/labinvest.2015.71

    Figure Lengend Snippet: FBXW7 promotes the ubiquitination of ENO1. ( a ) HEK293T cells were co-transfected with Myc-ubiquitin and wild-type FBXW7, FBXW7ΔF or sh FBXW7 B and then treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-Myc antibody and

    Article Snippet: Cells were treated with 10 µM MG132 or 25 µM GSK3β inhibitor VIII and incubated for 6 h. Antibodies against FBXW7 (ab109617) were purchased from Abcam.

    Techniques: Transfection, Immunoprecipitation

    Proteasome subunit up-regulation in β-thalassemia is Nrf1 dependent. (A-B) Wild-type murine fetal liver erythroid cultures were treated for 24 hours with 1μM sulforaphane or 0.1μM MG132 to activate Nrf2 or Nrf1, respectively. (A)

    Journal: Blood

    Article Title: Integrated protein quality-control pathways regulate free ?-globin in murine ?-thalassemia

    doi: 10.1182/blood-2011-12-397729

    Figure Lengend Snippet: Proteasome subunit up-regulation in β-thalassemia is Nrf1 dependent. (A-B) Wild-type murine fetal liver erythroid cultures were treated for 24 hours with 1μM sulforaphane or 0.1μM MG132 to activate Nrf2 or Nrf1, respectively. (A)

    Article Snippet: For Nrf1 and Nrf2 induction experiments, cells in differentiation medium were treated for 24 hours with 0.1μM MG132 or 1μM R-sulforaphane (LKT laboratories).

    Techniques: