mg132  (Millipore)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    R MG132
    Description:

    Catalog Number:
    M8699
    Price:
    None
    Applications:
    (R)-MG132 has been used in ubiquitination assay and is used as a proteasome inhibitor.
    Buy from Supplier


    Structured Review

    Millipore mg132
    R MG132

    https://www.bioz.com/result/mg132/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mg132 - by Bioz Stars, 2021-07
    99/100 stars

    Images

    1) Product Images from "Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels"

    Article Title: Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    Journal: Scientific Reports

    doi: 10.1038/srep40825

    Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM MG132 (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p
    Figure Legend Snippet: Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM MG132 (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p

    Techniques Used: Inhibition, Expressing, Molecular Weight, Modification, Western Blot

    Pharmacological suppression of proteasomal and lysosomal degradations alters subcellular localization of rEag1 in HEK293T cells. Representative confocal micrographs showing rEag1 immunofluorescence signals (green) in response to Cul7 co-expression ( A , B , C ), or 6-hour MG132 ( D , E , F ) or chloroquine ( G , H , I ) treatment. rEag1 and Myc-Cul7 were detected with anti-rEag1 and anti-Myc antibodies, respectively. Nuclei were counterstained with DAPI (blue). To verify plasma membrane localization, some cells were co-transfected with the DsRed-membrane expression vector (DsRed-Mem). ER and lysosomal localizations were detected by specific antibodies for the ER marker calnexin and the lysosome marker lamp1, respectively. Merge images are shown in the third column of each panel. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. See Supplementary Methods and Supplementary Figure S2B for further quantitative analyses. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least 3 independent experiments.
    Figure Legend Snippet: Pharmacological suppression of proteasomal and lysosomal degradations alters subcellular localization of rEag1 in HEK293T cells. Representative confocal micrographs showing rEag1 immunofluorescence signals (green) in response to Cul7 co-expression ( A , B , C ), or 6-hour MG132 ( D , E , F ) or chloroquine ( G , H , I ) treatment. rEag1 and Myc-Cul7 were detected with anti-rEag1 and anti-Myc antibodies, respectively. Nuclei were counterstained with DAPI (blue). To verify plasma membrane localization, some cells were co-transfected with the DsRed-membrane expression vector (DsRed-Mem). ER and lysosomal localizations were detected by specific antibodies for the ER marker calnexin and the lysosome marker lamp1, respectively. Merge images are shown in the third column of each panel. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. See Supplementary Methods and Supplementary Figure S2B for further quantitative analyses. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least 3 independent experiments.

    Techniques Used: Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Marker, Staining

    2) Product Images from "Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *"

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.299990

    Ataxin-3 targets mutant SOD1 to aggresomes to with autophagic activity. A , 293 cells co-expressing RFP-tagged SOD1 G85R , EGFP-tagged ataxin-3 and Flag-tagged p62 were incubated in 10 μ m MG132 for 12 h. The cells were then fixed and stained with
    Figure Legend Snippet: Ataxin-3 targets mutant SOD1 to aggresomes to with autophagic activity. A , 293 cells co-expressing RFP-tagged SOD1 G85R , EGFP-tagged ataxin-3 and Flag-tagged p62 were incubated in 10 μ m MG132 for 12 h. The cells were then fixed and stained with

    Techniques Used: Mutagenesis, Activity Assay, Expressing, Incubation, Staining

    Colocalization of mutant SOD1 and ataxin-3 in aggresomes. A , 293 cells were transfected with EGFP or SOD1 G85R -EGFP and treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were immunoprecipitated with rabbit polyclonal antibodies
    Figure Legend Snippet: Colocalization of mutant SOD1 and ataxin-3 in aggresomes. A , 293 cells were transfected with EGFP or SOD1 G85R -EGFP and treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were immunoprecipitated with rabbit polyclonal antibodies

    Techniques Used: Mutagenesis, Transfection, Immunoprecipitation

    Ataxin-3-mediated aggresome formation by mutant SOD1 depends on ataxin-3 DUB activity. A , 293 cells were co-transfected with SOD1 G85R -EGFP and HA or HA-ataxin-3, then treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were
    Figure Legend Snippet: Ataxin-3-mediated aggresome formation by mutant SOD1 depends on ataxin-3 DUB activity. A , 293 cells were co-transfected with SOD1 G85R -EGFP and HA or HA-ataxin-3, then treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were

    Techniques Used: Mutagenesis, Activity Assay, Transfection

    3) Product Images from "Inhibition of Proteasomal Activity Causes Inclusion Formation in Neuronal and Non-Neuronal Cells Overexpressing Parkin"

    Article Title: Inhibition of Proteasomal Activity Causes Inclusion Formation in Neuronal and Non-Neuronal Cells Overexpressing Parkin

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-02-0078

    Effects of truncation and point mutations of Parkin on inclusion body formation. (A) Schematic illustrating the positions of the point mutations and truncations of Parkin constructs used in B. Φ, AR-JP–associated mutation. (B) COS-7 cells were transfected with FLAG-Parkin or deletion/mutation constructs and cultured in the absence of MG132. The expression of Parkin was analyzed by immunofluorescence with anti-FLAG antibodies. Cells were scored for the presence of inclusion bodies. Error bars indicate the SE from the mean. Asterisk(s) indicate a significant difference between the number of inclusions observed with each construct compared with wild type, FLAG-Parkin, without addition of MG132. * p
    Figure Legend Snippet: Effects of truncation and point mutations of Parkin on inclusion body formation. (A) Schematic illustrating the positions of the point mutations and truncations of Parkin constructs used in B. Φ, AR-JP–associated mutation. (B) COS-7 cells were transfected with FLAG-Parkin or deletion/mutation constructs and cultured in the absence of MG132. The expression of Parkin was analyzed by immunofluorescence with anti-FLAG antibodies. Cells were scored for the presence of inclusion bodies. Error bars indicate the SE from the mean. Asterisk(s) indicate a significant difference between the number of inclusions observed with each construct compared with wild type, FLAG-Parkin, without addition of MG132. * p

    Techniques Used: Construct, Mutagenesis, Transfection, Cell Culture, Expressing, Immunofluorescence

    Effect of nocodazole on Parkin and PS-1 inclusions. COS-7 cells were transfected with FLAG-Parkin (A–L) or PS1 (M–X) constructs and grown in the absence (A–C and M–O) or presence of 20 μM MG132 (D–F and P–R) or 10 μg/ml nocodazole (G–I and S–U) or both (J–L and V–X) for 16 h. After methanol fixation, cells were probed with affinity-purified rabbit anti-Parkin peptide (1:50) (A–L; red), or affinity-purified rabbit anti-PS1 peptide (1:50) (M–X; red) antibodies, and rat anti-α-tubulin (1:500) (A–X; green) antibodies. Overlays of each set of three include 4,6-diamidino-2-phenylindole staining (blue) to identify nuclei (C, F, I, L, O, R, U, and X). Regions of colocalization within cells stain yellow. Arrowheads indicate Parkin inclusions; arrows indicate PS1 aggresomes. Bars, 20 μm.
    Figure Legend Snippet: Effect of nocodazole on Parkin and PS-1 inclusions. COS-7 cells were transfected with FLAG-Parkin (A–L) or PS1 (M–X) constructs and grown in the absence (A–C and M–O) or presence of 20 μM MG132 (D–F and P–R) or 10 μg/ml nocodazole (G–I and S–U) or both (J–L and V–X) for 16 h. After methanol fixation, cells were probed with affinity-purified rabbit anti-Parkin peptide (1:50) (A–L; red), or affinity-purified rabbit anti-PS1 peptide (1:50) (M–X; red) antibodies, and rat anti-α-tubulin (1:500) (A–X; green) antibodies. Overlays of each set of three include 4,6-diamidino-2-phenylindole staining (blue) to identify nuclei (C, F, I, L, O, R, U, and X). Regions of colocalization within cells stain yellow. Arrowheads indicate Parkin inclusions; arrows indicate PS1 aggresomes. Bars, 20 μm.

    Techniques Used: Transfection, Construct, Affinity Purification, Staining

    MG132 induces the formation of insoluble, high-molecular-weight Parkin species. COS-7 cells were transfected with FLAG-Parkin (left), FLAG-HHARI (right), or mock-transfected (middle) and grown in the presence or absence of MG132 for 16 or 28 h post-transfection. Cell lysates were prepared in buffer as indicated, and 10 μg of each soluble (S) and insoluble (I) fractions was separated by SDS-PAGE and analyzed by Western blotting. Blots were probed with mouse anti-FLAG (top), affinity-purified rabbit anti-UbcH7 peptide (1:5000) (middle), and anti-β-actin (bottom) antibodies. Arrows indicate the presence of each respective FLAG construct.
    Figure Legend Snippet: MG132 induces the formation of insoluble, high-molecular-weight Parkin species. COS-7 cells were transfected with FLAG-Parkin (left), FLAG-HHARI (right), or mock-transfected (middle) and grown in the presence or absence of MG132 for 16 or 28 h post-transfection. Cell lysates were prepared in buffer as indicated, and 10 μg of each soluble (S) and insoluble (I) fractions was separated by SDS-PAGE and analyzed by Western blotting. Blots were probed with mouse anti-FLAG (top), affinity-purified rabbit anti-UbcH7 peptide (1:5000) (middle), and anti-β-actin (bottom) antibodies. Arrows indicate the presence of each respective FLAG construct.

    Techniques Used: Molecular Weight, Transfection, SDS Page, Western Blot, Affinity Purification, Construct

    Parkin inclusion bodies display aggresome-like characteristics. (A–H) Parkin inclusions cause disruption of γ-tubulin localization. COS-7 cells were transfected with FLAG-Parkin (A–C and E–G) in the presence (E–H) or absence (A–D) of MG132. Cells were stained with affinity-purified rabbit anti-Parkin (red) and mouse γ-tubulin (1:1000) (green) antibodies. Staining of untransfected cells confirmed aggregation of γ-tubulin only occurs in cells overexpressing Parkin (D and H). Red staining in D and H represents 4,6-diamidino-2-phenylindole staining of the nucleus. Arrowheads indicate γ-tubulin localization. (I–N) Partial colocalization of Parkin and vimentin near the centrosome. Cells overexpressing Parkin were stained with affinity-purified rabbit anti-Parkin (red) and anti-vimentin (1:40) antibodies (green) in the presence (L–N) or absence (I–K) of MG132. Arrow indicates region of colocalization in N. (O–Q) Acetylated α-tubulin localizes to Parkin inclusions. Cells overexpressing Parkin were stained with affinity-purified rabbit anti-Parkin (red) and mouse anti-acetylated α-tubulin (1:2000) antibodies (green). Panels show untreated (O), MG132-treated (P), or combined MG132 and nocodazole-treated (Q) cells. Arrows indicate colocalization within the inclusion in P and Q. The asterisk ( * ) indicates an untransfected cell with intact acetylated α-tubulin staining in Q. Overlays of each set include 4,6-diamidino-2-phenylindole staining (blue) (C, G, K, and N) to identify the nucleus. Mitochondria localize with Parkin inclusions. In R and S, Parkin expression is indicated by the green fluorescence and the mitochondria were labeled using MitoTracker (used at 300 nM; red fluorescence). With the exception of (R and S), which were fixed with 4% (wt/vol) paraformaldehyde, all cells were fixed with methanol. Bars, 20 μm.
    Figure Legend Snippet: Parkin inclusion bodies display aggresome-like characteristics. (A–H) Parkin inclusions cause disruption of γ-tubulin localization. COS-7 cells were transfected with FLAG-Parkin (A–C and E–G) in the presence (E–H) or absence (A–D) of MG132. Cells were stained with affinity-purified rabbit anti-Parkin (red) and mouse γ-tubulin (1:1000) (green) antibodies. Staining of untransfected cells confirmed aggregation of γ-tubulin only occurs in cells overexpressing Parkin (D and H). Red staining in D and H represents 4,6-diamidino-2-phenylindole staining of the nucleus. Arrowheads indicate γ-tubulin localization. (I–N) Partial colocalization of Parkin and vimentin near the centrosome. Cells overexpressing Parkin were stained with affinity-purified rabbit anti-Parkin (red) and anti-vimentin (1:40) antibodies (green) in the presence (L–N) or absence (I–K) of MG132. Arrow indicates region of colocalization in N. (O–Q) Acetylated α-tubulin localizes to Parkin inclusions. Cells overexpressing Parkin were stained with affinity-purified rabbit anti-Parkin (red) and mouse anti-acetylated α-tubulin (1:2000) antibodies (green). Panels show untreated (O), MG132-treated (P), or combined MG132 and nocodazole-treated (Q) cells. Arrows indicate colocalization within the inclusion in P and Q. The asterisk ( * ) indicates an untransfected cell with intact acetylated α-tubulin staining in Q. Overlays of each set include 4,6-diamidino-2-phenylindole staining (blue) (C, G, K, and N) to identify the nucleus. Mitochondria localize with Parkin inclusions. In R and S, Parkin expression is indicated by the green fluorescence and the mitochondria were labeled using MitoTracker (used at 300 nM; red fluorescence). With the exception of (R and S), which were fixed with 4% (wt/vol) paraformaldehyde, all cells were fixed with methanol. Bars, 20 μm.

    Techniques Used: Transfection, Staining, Affinity Purification, Expressing, Fluorescence, Labeling

    4) Product Images from "Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity"

    Article Title: Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305049

    Proteasome, phosphatase inhibitor, and APC/C deletions restore securin levels. (A and B) Western blot (20 oocytes/lane; A) and analysis of securin (B) in oocytes during GV arrest, meiosis I (MI), and meiosis I after incubation with roscovitine (R), UO126 (U), MG132, or okadaic acid (OA). Results are compared with the meiosis I lane, which is set at 100%. (C and D) Western blot (20 oocytes/lane, n = 2; C) and analysis of securin (D) in Apc2 -deleted ( Apc2 −/− ) and control ( Apc2 +/+ ) oocytes at meiosis I in the presence or absence of roscovitine and UO126. Error bars show SDs. **, P
    Figure Legend Snippet: Proteasome, phosphatase inhibitor, and APC/C deletions restore securin levels. (A and B) Western blot (20 oocytes/lane; A) and analysis of securin (B) in oocytes during GV arrest, meiosis I (MI), and meiosis I after incubation with roscovitine (R), UO126 (U), MG132, or okadaic acid (OA). Results are compared with the meiosis I lane, which is set at 100%. (C and D) Western blot (20 oocytes/lane, n = 2; C) and analysis of securin (D) in Apc2 -deleted ( Apc2 −/− ) and control ( Apc2 +/+ ) oocytes at meiosis I in the presence or absence of roscovitine and UO126. Error bars show SDs. **, P

    Techniques Used: Western Blot, Incubation

    5) Product Images from "RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells ▿RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells ▿ †"

    Article Title: RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells ▿RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01778-08

    IE2 activation of the CMV promoter is enhanced by siPML and siUBC9 but inhibited by MG132. (A) siRNAs against pml and sumo1 genes were transfected into Vero E6 cells, followed by vAcIE2+vAcL cotransduction. A luciferase assay detected enhanced effects from both siRNAs on IE2 trans -activation of the CMV promoter. siGFP (against egfp ) was used as a negative control, while siLUC (against luciferase ) was a positive control for siRNA efficiency. Data are presented as percentages of RLU in the absence of siRNA ( n = 4). * and **, P
    Figure Legend Snippet: IE2 activation of the CMV promoter is enhanced by siPML and siUBC9 but inhibited by MG132. (A) siRNAs against pml and sumo1 genes were transfected into Vero E6 cells, followed by vAcIE2+vAcL cotransduction. A luciferase assay detected enhanced effects from both siRNAs on IE2 trans -activation of the CMV promoter. siGFP (against egfp ) was used as a negative control, while siLUC (against luciferase ) was a positive control for siRNA efficiency. Data are presented as percentages of RLU in the absence of siRNA ( n = 4). * and **, P

    Techniques Used: Activation Assay, Transfection, Luciferase, Negative Control, Positive Control

    6) Product Images from "Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice"

    Article Title: Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01387-6

    Identification of ubiquitinated DPPA3. a Schematic of procedures to identify ubiquitin-regulated maternal proteins. b Immunoblot with anti-ubiquitin antibody of poly-ubiquitinated maternal proteins isolated by agarose-TUBE before or after treatment with EPO. Molecular masses (kDa) on left. Ub, ubiquitin. c Dot plot of the ratio of identified maternal proteins before (WT) and after (EPO+) treatment with EPO as determined by stable isotope labeling and microscale tandem mass spectrometry. d Immunoblot of DPPA3 in ovulated MII eggs (E) and fertilized 2C embryos (2C) with anti-DPPA3 antibodies. Staining with anti-ubiquitin and anti-tubulin antibodies were used as load controls. Molecular masses (kDa) on right. e Same as d but with ovulated MII eggs (E) and parthenogenetically activated 2C embryos (a2C). f Immunoblot with anti-DPPA3 antibody of 2C lysates isolated using agarose-TUBE before or after treatment with MG132, a proteasome inhibitor. Staining with anti-ubiquitin and anti-tubulin antibodies were used as load controls. Molecular masses (kDa) on right
    Figure Legend Snippet: Identification of ubiquitinated DPPA3. a Schematic of procedures to identify ubiquitin-regulated maternal proteins. b Immunoblot with anti-ubiquitin antibody of poly-ubiquitinated maternal proteins isolated by agarose-TUBE before or after treatment with EPO. Molecular masses (kDa) on left. Ub, ubiquitin. c Dot plot of the ratio of identified maternal proteins before (WT) and after (EPO+) treatment with EPO as determined by stable isotope labeling and microscale tandem mass spectrometry. d Immunoblot of DPPA3 in ovulated MII eggs (E) and fertilized 2C embryos (2C) with anti-DPPA3 antibodies. Staining with anti-ubiquitin and anti-tubulin antibodies were used as load controls. Molecular masses (kDa) on right. e Same as d but with ovulated MII eggs (E) and parthenogenetically activated 2C embryos (a2C). f Immunoblot with anti-DPPA3 antibody of 2C lysates isolated using agarose-TUBE before or after treatment with MG132, a proteasome inhibitor. Staining with anti-ubiquitin and anti-tubulin antibodies were used as load controls. Molecular masses (kDa) on right

    Techniques Used: Isolation, Labeling, Mass Spectrometry, Staining

    Partial cleavage of maternal DPPA3. a CBB staining of SDS-PAGE (left) and immunoblot with anti-DPPA3 antibody (right) of partially cleaved DPPA3 after in vitro digestion using recombinant DPPA3 and 20S proteasome purified from mouse ovaries in the presence and absence of MG132. Molecular masses (kDa) on left. b Schematics of cRNA encoding DPPA3 WT (top) or DPPA3 R60A (bottom) fused at their C-termini to mVenus that were microinjected into 1C zygotes. c cRNA encoding DPPA3 WT (left) and DPPA3 R60A (right) was injection into 1C zygotes and cultured to 2C, and 4C embryos prior to imaging with confocal microscopy alone and merged with DIC/Hoechst-stained images. Scale bar, 20 μm. d Immunoblots with anti-DPPA3 antibodies of embryo extracts from Dppa3 WT before (left) and after (right) treatment with MG132 and of embryo extracts from Dppa3 R60A (middle) mice. Staining with anti-tubulin antibodies was used as a load control. e Ovulated MII egg; 2C, fertilized 2C embryo; a2C, parthenogenetically activated 2C embryo. Molecular masses (kDa) on right. e Percentage of embryos at specified stage after parthenogenetic activation of eggs derived from Dppa3 R60A/+ and Dppa3 R60A/− female mice. Error bars, s.d. from three replicates, *** Ρ
    Figure Legend Snippet: Partial cleavage of maternal DPPA3. a CBB staining of SDS-PAGE (left) and immunoblot with anti-DPPA3 antibody (right) of partially cleaved DPPA3 after in vitro digestion using recombinant DPPA3 and 20S proteasome purified from mouse ovaries in the presence and absence of MG132. Molecular masses (kDa) on left. b Schematics of cRNA encoding DPPA3 WT (top) or DPPA3 R60A (bottom) fused at their C-termini to mVenus that were microinjected into 1C zygotes. c cRNA encoding DPPA3 WT (left) and DPPA3 R60A (right) was injection into 1C zygotes and cultured to 2C, and 4C embryos prior to imaging with confocal microscopy alone and merged with DIC/Hoechst-stained images. Scale bar, 20 μm. d Immunoblots with anti-DPPA3 antibodies of embryo extracts from Dppa3 WT before (left) and after (right) treatment with MG132 and of embryo extracts from Dppa3 R60A (middle) mice. Staining with anti-tubulin antibodies was used as a load control. e Ovulated MII egg; 2C, fertilized 2C embryo; a2C, parthenogenetically activated 2C embryo. Molecular masses (kDa) on right. e Percentage of embryos at specified stage after parthenogenetic activation of eggs derived from Dppa3 R60A/+ and Dppa3 R60A/− female mice. Error bars, s.d. from three replicates, *** Ρ

    Techniques Used: Staining, SDS Page, In Vitro, Recombinant, Purification, Injection, Cell Culture, Imaging, Confocal Microscopy, Western Blot, Mouse Assay, Activation Assay, Derivative Assay

    7) Product Images from "Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells"

    Article Title: Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2015.154

    GSK3β-mediated proteasomal degradation of β-catenin by silymarin treatment. (A, B) HCT116 and SW480 cells were pretreated with 10 μM of MG132 or 20 μM of SB216763 for 2 h and then co-treated with 100 μM of silymarin for 6 h. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (C) HCT116 cells were treated with 100 μM of silymarin for the indicated times. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for phospho-GSK3β and total-GSK3β. * p
    Figure Legend Snippet: GSK3β-mediated proteasomal degradation of β-catenin by silymarin treatment. (A, B) HCT116 and SW480 cells were pretreated with 10 μM of MG132 or 20 μM of SB216763 for 2 h and then co-treated with 100 μM of silymarin for 6 h. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (C) HCT116 cells were treated with 100 μM of silymarin for the indicated times. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for phospho-GSK3β and total-GSK3β. * p

    Techniques Used: SDS Page, Western Blot

    8) Product Images from "Arsenic decreases RXR?-dependent transcription of CYP3A and suppresses immune regulators in hepatocytes"

    Article Title: Arsenic decreases RXR?-dependent transcription of CYP3A and suppresses immune regulators in hepatocytes

    Journal: International Immunopharmacology

    doi: 10.1016/j.intimp.2012.01.008

    Effects of proteasomal inhibition on RXRα protein. (A) Primary human hepatocyte cultures were untreated (Ctl) or treated with MG132 (MG) (5 μM) for 6 hr. Whole cells were treated with NEM (5 mM) at the time of lysis, and 20 μg
    Figure Legend Snippet: Effects of proteasomal inhibition on RXRα protein. (A) Primary human hepatocyte cultures were untreated (Ctl) or treated with MG132 (MG) (5 μM) for 6 hr. Whole cells were treated with NEM (5 mM) at the time of lysis, and 20 μg

    Techniques Used: Inhibition, CTL Assay, Lysis

    9) Product Images from "Tamoxifen Inhibits ER-negative Breast Cancer Cell Invasion and Metastasis by Accelerating Twist1 Degradation"

    Article Title: Tamoxifen Inhibits ER-negative Breast Cancer Cell Invasion and Metastasis by Accelerating Twist1 Degradation

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.11380

    Tamoxifen-accelerated Twist1 degradation is dependent on the proteasome pathway, but is independent of estrogen and MAPK signaling pathways. A. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression (HEK293+Twist1) after cells were treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. HEK293 parent cells without Twist1 expression served as a negative control. B. Western blot analysis of Twist1 in 168FARN cells treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen. C. Western blot analysis of Twist1 in HEK293+Twist1, 4T1 and 168FARN cells treated for 24 hours with vehicle (V, ethanol), or 10 μM of 17β-estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (4OH) and tamoxifen (Tam) as indicated. D. Western blot analysis of active phospho-Erk1/2, total Erk1/2, active phospho-p38, total p38, active phospho-JNK, and total JNK in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. E. Western blot analysis of phospho-Ser68-Twist1 and total Twist1 in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. Results shown in all panels are representative results of at least three repeat assays.
    Figure Legend Snippet: Tamoxifen-accelerated Twist1 degradation is dependent on the proteasome pathway, but is independent of estrogen and MAPK signaling pathways. A. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression (HEK293+Twist1) after cells were treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. HEK293 parent cells without Twist1 expression served as a negative control. B. Western blot analysis of Twist1 in 168FARN cells treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen. C. Western blot analysis of Twist1 in HEK293+Twist1, 4T1 and 168FARN cells treated for 24 hours with vehicle (V, ethanol), or 10 μM of 17β-estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (4OH) and tamoxifen (Tam) as indicated. D. Western blot analysis of active phospho-Erk1/2, total Erk1/2, active phospho-p38, total p38, active phospho-JNK, and total JNK in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. E. Western blot analysis of phospho-Ser68-Twist1 and total Twist1 in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. Results shown in all panels are representative results of at least three repeat assays.

    Techniques Used: Western Blot, Expressing, Negative Control

    10) Product Images from "Inducible Nitric-oxide Synthase and Nitric Oxide Donor Decrease Insulin Receptor Substrate-2 Protein Expression by Promoting Proteasome-dependent Degradation in Pancreatic ?-Cells"

    Article Title: Inducible Nitric-oxide Synthase and Nitric Oxide Donor Decrease Insulin Receptor Substrate-2 Protein Expression by Promoting Proteasome-dependent Degradation in Pancreatic ?-Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.192732

    Proteasome-dependent protein degradation of IRS-2 in NO donor-treated β-cells. INS-1/832 cells were treated with and without GSNO (200 μ m ) up to 4 h in the presence and absence of proteasome inhibitors, MG132 (1 μ m ) and lactacystin
    Figure Legend Snippet: Proteasome-dependent protein degradation of IRS-2 in NO donor-treated β-cells. INS-1/832 cells were treated with and without GSNO (200 μ m ) up to 4 h in the presence and absence of proteasome inhibitors, MG132 (1 μ m ) and lactacystin

    Techniques Used:

    11) Product Images from "Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis"

    Article Title: Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis

    Journal: Molecular Cancer

    doi: 10.1186/s12943-016-0543-1

    ITSN-1s enhances Cbl-Eps8 interaction leading increased ubiquitination of Eps8. a A549 and A549 + ITSN-1s cells pretreated with MG132 (10 μM for 2 h) and untreated cells were lysed and subjected to IP with Eps8 Ab followed by WB with Eps8 and Ub Abs. Arrows and bracket show ubiquitinated Eps8. b Densitometry of Ub-Eps8 expressed as percentage of control. c IP with Cbl Ab followed by WB with Eps8 Ab. The lower panels illustrate the Cbl and Eps8 protein levels in A549 and A549 + ITSN-1s cell lysates. Rabbit IgG was used as control (not shown). d Densitometry of Eps8-Cbl interaction expressed as percentage of control. e A549 and A549 + ITSN-1s cells transfected with Cbl-siRNA#1 and #2 or control-siRNA. Lysates were blotted with Cbl Ab (middle panel) followed by Eps8 Ab ( upper panel ). Actin was used as loading control ( lower panel ). Data are shown as mean ± SE. * p
    Figure Legend Snippet: ITSN-1s enhances Cbl-Eps8 interaction leading increased ubiquitination of Eps8. a A549 and A549 + ITSN-1s cells pretreated with MG132 (10 μM for 2 h) and untreated cells were lysed and subjected to IP with Eps8 Ab followed by WB with Eps8 and Ub Abs. Arrows and bracket show ubiquitinated Eps8. b Densitometry of Ub-Eps8 expressed as percentage of control. c IP with Cbl Ab followed by WB with Eps8 Ab. The lower panels illustrate the Cbl and Eps8 protein levels in A549 and A549 + ITSN-1s cell lysates. Rabbit IgG was used as control (not shown). d Densitometry of Eps8-Cbl interaction expressed as percentage of control. e A549 and A549 + ITSN-1s cells transfected with Cbl-siRNA#1 and #2 or control-siRNA. Lysates were blotted with Cbl Ab (middle panel) followed by Eps8 Ab ( upper panel ). Actin was used as loading control ( lower panel ). Data are shown as mean ± SE. * p

    Techniques Used: Western Blot, Transfection

    12) Product Images from "Rotavirus Replication Requires a Functional Proteasome for Effective Assembly of Viroplasms ▿"

    Article Title: Rotavirus Replication Requires a Functional Proteasome for Effective Assembly of Viroplasms ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01631-10

    Effect of proteasome inhibition on viral polymerase activities and production of viral proteins. (A) Transcriptase activity of DLPs. The plot shows the incorporation of [α- 32 P]UTP into newly synthesized RNA from DLPs treated with DMSO or MG132
    Figure Legend Snippet: Effect of proteasome inhibition on viral polymerase activities and production of viral proteins. (A) Transcriptase activity of DLPs. The plot shows the incorporation of [α- 32 P]UTP into newly synthesized RNA from DLPs treated with DMSO or MG132

    Techniques Used: Inhibition, Activity Assay, Synthesized

    Kinetics of rotavirus infection and proteasome inhibition. Western blots of MA104 cells infected with OSU (MOI, 3) and treated with MG132 (10 μM) for different times, as indicated. *, specific time point at which cell lysates were prepared.
    Figure Legend Snippet: Kinetics of rotavirus infection and proteasome inhibition. Western blots of MA104 cells infected with OSU (MOI, 3) and treated with MG132 (10 μM) for different times, as indicated. *, specific time point at which cell lysates were prepared.

    Techniques Used: Infection, Inhibition, Western Blot

    Proteasome inhibition and IFN-β induction. (A) Schematic representation of the experiments shown in B and C, performed on virus-infected MA104 cells. (B) Western blot showing IRF3 levels in extracts from MG132-treated and nontreated cells. Blots
    Figure Legend Snippet: Proteasome inhibition and IFN-β induction. (A) Schematic representation of the experiments shown in B and C, performed on virus-infected MA104 cells. (B) Western blot showing IRF3 levels in extracts from MG132-treated and nontreated cells. Blots

    Techniques Used: Inhibition, Infection, Western Blot

    Time window treatment with proteasome inhibitors. (A) Scheme of the experiment performed with MA104 cells exposed to virus (OSU; MOI, 3) for 1 h and analyzed at the starting point and endpoint of the indicated time window treatments with DMSO, MG132,
    Figure Legend Snippet: Time window treatment with proteasome inhibitors. (A) Scheme of the experiment performed with MA104 cells exposed to virus (OSU; MOI, 3) for 1 h and analyzed at the starting point and endpoint of the indicated time window treatments with DMSO, MG132,

    Techniques Used:

    Effect of proteasome inhibition on different rotavirus strains. Western blot of cellular extracts obtained from OSU- and SA11-infected MA104 cells treated or not treated (No Inf.) with MG132 (10 μM) from 1 h to 7 h p.i. Blots were reacted with
    Figure Legend Snippet: Effect of proteasome inhibition on different rotavirus strains. Western blot of cellular extracts obtained from OSU- and SA11-infected MA104 cells treated or not treated (No Inf.) with MG132 (10 μM) from 1 h to 7 h p.i. Blots were reacted with

    Techniques Used: Inhibition, Western Blot, Infection

    Effect of proteasome activity on viroplasm formation. (A) Fluorescence analysis of viroplasm formation on NSP5-EGFP cells infected with rotavirus (OSU; MOI, 3) and treated or not treated with MG132 (10 μM) or bortezomib (10 μM) at different
    Figure Legend Snippet: Effect of proteasome activity on viroplasm formation. (A) Fluorescence analysis of viroplasm formation on NSP5-EGFP cells infected with rotavirus (OSU; MOI, 3) and treated or not treated with MG132 (10 μM) or bortezomib (10 μM) at different

    Techniques Used: Activity Assay, Fluorescence, Infection

    13) Product Images from "Selenate Prevents Adipogenesis through Induction of Selenoprotein S and Attenuation of Endoplasmic Reticulum Stress"

    Article Title: Selenate Prevents Adipogenesis through Induction of Selenoprotein S and Attenuation of Endoplasmic Reticulum Stress

    Journal: Molecules

    doi: 10.3390/molecules23112882

    Regulation of SEPS1 by selenate and adipogenic cocktail treatments. ( A ) After 24 h pretreatment of one-day post-confluent 3T3-L1 preadipocytes with 50 µM selenate, the cells were differentiated in individual components of the adipogenic cocktail, dexamethasone (D), isobutylmethylxanthine (M), and insulin (I), and DMI. Protein samples were collected on Day 2 (D2) and were subjected to immunoblot analysis. ( B ) The samples shown in D0 are one-day post-confluent 3T3-L1 preadipocytes added in the presence or absence of selenate and MG132 for 24 h. The samples shown in D1 include 3T3-L1 preadipocytes treated with DMI with or without MG132 for 24 h, after selenate pretreatment for 24 h. The protein samples at D0 and D1 were subjected to immunoblot assay.
    Figure Legend Snippet: Regulation of SEPS1 by selenate and adipogenic cocktail treatments. ( A ) After 24 h pretreatment of one-day post-confluent 3T3-L1 preadipocytes with 50 µM selenate, the cells were differentiated in individual components of the adipogenic cocktail, dexamethasone (D), isobutylmethylxanthine (M), and insulin (I), and DMI. Protein samples were collected on Day 2 (D2) and were subjected to immunoblot analysis. ( B ) The samples shown in D0 are one-day post-confluent 3T3-L1 preadipocytes added in the presence or absence of selenate and MG132 for 24 h. The samples shown in D1 include 3T3-L1 preadipocytes treated with DMI with or without MG132 for 24 h, after selenate pretreatment for 24 h. The protein samples at D0 and D1 were subjected to immunoblot assay.

    Techniques Used:

    14) Product Images from "Targeted Destruction of DNA Replication Protein Cdc6 by Cell Death Pathways in Mammals and Yeast"

    Article Title: Targeted Destruction of DNA Replication Protein Cdc6 by Cell Death Pathways in Mammals and Yeast

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.02-02-0010

    Adozelesin induces ubiquitination and proteasome-dependent degradation of Cdc6. (A) MDA cells were treated with 40 nM adozelesin (Ado) for the indicated times in presence or absence of 10 μM MG132 (MG), 10 μg/ml cycloheximide (CHX), or combinations as indicated. Cell extracts were analyzed for Cdc6, Cdc25A, or MCM2 levels by immunoblotting. (B) MCF-7 cells were transfected with vectors expressing Myc-Cdc6 wild-type [Myc-Cdc6 (wt)] or mutated destruction box [Myc-Cdc6(Δ)] or KEN motif [Myc-Cdc6(KEN)] proteins. Cells were treated 6 h with adozelesin (Ado), cycloheximide (CHX), or MG beginning 24 h after transfection and analyzed for the level of transfected Cdc6 by immunoblotting with anti-Myc antibodies. Signals from immunoblots in three independent experiments were quantitated by densitometry and the averages and SEs presented above each lane. (C) MCF-7 cells were cotransfected with expression vectors for HA-tagged ubiquitin and the Myc-tagged wild-type and mutant Cdc6 described above. Lysates were made from cells after adozelesin treatment for 6 h beginning 24 h after transfection, and Myc-tagged Cdc6 proteins were immunoprecipitated with Myc antibodies. Immunoprecipitated proteins were analyzed for HA-ubiquitin and then for Myc-tagged Cdc6 by immunoblotting with anti-HA (top) and anti-Myc (bottom) antibodies. hIg is the IgG light chain that reacts with the secondary antibody.
    Figure Legend Snippet: Adozelesin induces ubiquitination and proteasome-dependent degradation of Cdc6. (A) MDA cells were treated with 40 nM adozelesin (Ado) for the indicated times in presence or absence of 10 μM MG132 (MG), 10 μg/ml cycloheximide (CHX), or combinations as indicated. Cell extracts were analyzed for Cdc6, Cdc25A, or MCM2 levels by immunoblotting. (B) MCF-7 cells were transfected with vectors expressing Myc-Cdc6 wild-type [Myc-Cdc6 (wt)] or mutated destruction box [Myc-Cdc6(Δ)] or KEN motif [Myc-Cdc6(KEN)] proteins. Cells were treated 6 h with adozelesin (Ado), cycloheximide (CHX), or MG beginning 24 h after transfection and analyzed for the level of transfected Cdc6 by immunoblotting with anti-Myc antibodies. Signals from immunoblots in three independent experiments were quantitated by densitometry and the averages and SEs presented above each lane. (C) MCF-7 cells were cotransfected with expression vectors for HA-tagged ubiquitin and the Myc-tagged wild-type and mutant Cdc6 described above. Lysates were made from cells after adozelesin treatment for 6 h beginning 24 h after transfection, and Myc-tagged Cdc6 proteins were immunoprecipitated with Myc antibodies. Immunoprecipitated proteins were analyzed for HA-ubiquitin and then for Myc-tagged Cdc6 by immunoblotting with anti-HA (top) and anti-Myc (bottom) antibodies. hIg is the IgG light chain that reacts with the secondary antibody.

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

    Cdc6 destruction is induced by UV radiation and TNF-α and coincides with a caspase-dependent apoptotic response. (A) MDA cells were irradiated (25 or 50 J/m 2 UV radiation) and returned to normal culture conditions in the presence or absence of 1 μM of the kinase inhibitor UCN-01 for 0–6 h. Proteins extracted at indicated times after irradiation were analyzed by immunoblotting. (B) MDA cells were treated with 1 μM UCN01, 40 nM adozelesin (Ado), or both (Ado+UCN01) for indicated times and extracted proteins analyzed by immunoblotting. (C) DNA was isolated from MDA cells treated with various combinations of Ado, 100 ng/ml TNF-α (TNF), and 10 μg/ml cycloheximide (TNF-CHX) with or without 10 μM MG132 (MG) or 10 μM caspase inhibitor I (CI) for indicated times and then size-fractionated on ethidium bromide-containing agarose gels. The standard (std) is a 100-base pair ladder. (D) Cells were treated with the indicated compounds for 0–6 h and protein extracts analyzed by immunoblotting.
    Figure Legend Snippet: Cdc6 destruction is induced by UV radiation and TNF-α and coincides with a caspase-dependent apoptotic response. (A) MDA cells were irradiated (25 or 50 J/m 2 UV radiation) and returned to normal culture conditions in the presence or absence of 1 μM of the kinase inhibitor UCN-01 for 0–6 h. Proteins extracted at indicated times after irradiation were analyzed by immunoblotting. (B) MDA cells were treated with 1 μM UCN01, 40 nM adozelesin (Ado), or both (Ado+UCN01) for indicated times and extracted proteins analyzed by immunoblotting. (C) DNA was isolated from MDA cells treated with various combinations of Ado, 100 ng/ml TNF-α (TNF), and 10 μg/ml cycloheximide (TNF-CHX) with or without 10 μM MG132 (MG) or 10 μM caspase inhibitor I (CI) for indicated times and then size-fractionated on ethidium bromide-containing agarose gels. The standard (std) is a 100-base pair ladder. (D) Cells were treated with the indicated compounds for 0–6 h and protein extracts analyzed by immunoblotting.

    Techniques Used: Multiple Displacement Amplification, Irradiation, Isolation

    Adozelesin induces the proteasome-dependent destruction of Cdc6 in budding yeast. (A and C) S. cerevisiae cells harboring plasmids expressing the S. cerevisiae Cdc6 (scCdc6) or Arabidopsis CDC6 (AtCDC6) genes regulated by a galactose-inducible promoter were changed from raffinose- to galactose-containing medium to induce the ectopic expression of scCdc6 or AtCDC6. They were treated with 4 μM adozelesin and/or 250 μM MG132 beginning 1 h (scCdc6 experiments) or 1.5 h (AtCDC6 experiments) after galactose induction. Total protein lysates made at the indicated times after induction were analyzed by immunoblotting for S. cerevisiae Cdc6 (scCdc6) or arabidopsis Cdc6 (AtCDC6) proteins, or for scMcm2 protein as a control. (B) cdc4 cells were synchronized in early S phase by release from an α factor G1 block into HU and then cultured in the continued presence of HU at the nonpermissive temperature (37°C) for 2 h to destroy the activity of Cdc4p. Cycloheximide and adozelesin were then added for the times indicated before cells were lysed for Western blot analysis. Arp, actin-related protein.
    Figure Legend Snippet: Adozelesin induces the proteasome-dependent destruction of Cdc6 in budding yeast. (A and C) S. cerevisiae cells harboring plasmids expressing the S. cerevisiae Cdc6 (scCdc6) or Arabidopsis CDC6 (AtCDC6) genes regulated by a galactose-inducible promoter were changed from raffinose- to galactose-containing medium to induce the ectopic expression of scCdc6 or AtCDC6. They were treated with 4 μM adozelesin and/or 250 μM MG132 beginning 1 h (scCdc6 experiments) or 1.5 h (AtCDC6 experiments) after galactose induction. Total protein lysates made at the indicated times after induction were analyzed by immunoblotting for S. cerevisiae Cdc6 (scCdc6) or arabidopsis Cdc6 (AtCDC6) proteins, or for scMcm2 protein as a control. (B) cdc4 cells were synchronized in early S phase by release from an α factor G1 block into HU and then cultured in the continued presence of HU at the nonpermissive temperature (37°C) for 2 h to destroy the activity of Cdc4p. Cycloheximide and adozelesin were then added for the times indicated before cells were lysed for Western blot analysis. Arp, actin-related protein.

    Techniques Used: Expressing, Blocking Assay, Cell Culture, Activity Assay, Western Blot

    15) Product Images from "Targeted Destruction of DNA Replication Protein Cdc6 by Cell Death Pathways in Mammals and Yeast"

    Article Title: Targeted Destruction of DNA Replication Protein Cdc6 by Cell Death Pathways in Mammals and Yeast

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.02-02-0010

    Adozelesin induces ubiquitination and proteasome-dependent degradation of Cdc6. (A) MDA cells were treated with 40 nM adozelesin (Ado) for the indicated times in presence or absence of 10 μM MG132 (MG), 10 μg/ml cycloheximide (CHX), or combinations as indicated. Cell extracts were analyzed for Cdc6, Cdc25A, or MCM2 levels by immunoblotting. (B) MCF-7 cells were transfected with vectors expressing Myc-Cdc6 wild-type [Myc-Cdc6 (wt)] or mutated destruction box [Myc-Cdc6(Δ)] or KEN motif [Myc-Cdc6(KEN)] proteins. Cells were treated 6 h with adozelesin (Ado), cycloheximide (CHX), or MG beginning 24 h after transfection and analyzed for the level of transfected Cdc6 by immunoblotting with anti-Myc antibodies. Signals from immunoblots in three independent experiments were quantitated by densitometry and the averages and SEs presented above each lane. (C) MCF-7 cells were cotransfected with expression vectors for HA-tagged ubiquitin and the Myc-tagged wild-type and mutant Cdc6 described above. Lysates were made from cells after adozelesin treatment for 6 h beginning 24 h after transfection, and Myc-tagged Cdc6 proteins were immunoprecipitated with Myc antibodies. Immunoprecipitated proteins were analyzed for HA-ubiquitin and then for Myc-tagged Cdc6 by immunoblotting with anti-HA (top) and anti-Myc (bottom) antibodies. hIg is the IgG light chain that reacts with the secondary antibody.
    Figure Legend Snippet: Adozelesin induces ubiquitination and proteasome-dependent degradation of Cdc6. (A) MDA cells were treated with 40 nM adozelesin (Ado) for the indicated times in presence or absence of 10 μM MG132 (MG), 10 μg/ml cycloheximide (CHX), or combinations as indicated. Cell extracts were analyzed for Cdc6, Cdc25A, or MCM2 levels by immunoblotting. (B) MCF-7 cells were transfected with vectors expressing Myc-Cdc6 wild-type [Myc-Cdc6 (wt)] or mutated destruction box [Myc-Cdc6(Δ)] or KEN motif [Myc-Cdc6(KEN)] proteins. Cells were treated 6 h with adozelesin (Ado), cycloheximide (CHX), or MG beginning 24 h after transfection and analyzed for the level of transfected Cdc6 by immunoblotting with anti-Myc antibodies. Signals from immunoblots in three independent experiments were quantitated by densitometry and the averages and SEs presented above each lane. (C) MCF-7 cells were cotransfected with expression vectors for HA-tagged ubiquitin and the Myc-tagged wild-type and mutant Cdc6 described above. Lysates were made from cells after adozelesin treatment for 6 h beginning 24 h after transfection, and Myc-tagged Cdc6 proteins were immunoprecipitated with Myc antibodies. Immunoprecipitated proteins were analyzed for HA-ubiquitin and then for Myc-tagged Cdc6 by immunoblotting with anti-HA (top) and anti-Myc (bottom) antibodies. hIg is the IgG light chain that reacts with the secondary antibody.

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

    Cdc6 destruction is induced by UV radiation and TNF-α and coincides with a caspase-dependent apoptotic response. (A) MDA cells were irradiated (25 or 50 J/m 2 UV radiation) and returned to normal culture conditions in the presence or absence of 1 μM of the kinase inhibitor UCN-01 for 0–6 h. Proteins extracted at indicated times after irradiation were analyzed by immunoblotting. (B) MDA cells were treated with 1 μM UCN01, 40 nM adozelesin (Ado), or both (Ado+UCN01) for indicated times and extracted proteins analyzed by immunoblotting. (C) DNA was isolated from MDA cells treated with various combinations of Ado, 100 ng/ml TNF-α (TNF), and 10 μg/ml cycloheximide (TNF-CHX) with or without 10 μM MG132 (MG) or 10 μM caspase inhibitor I (CI) for indicated times and then size-fractionated on ethidium bromide-containing agarose gels. The standard (std) is a 100-base pair ladder. (D) Cells were treated with the indicated compounds for 0–6 h and protein extracts analyzed by immunoblotting.
    Figure Legend Snippet: Cdc6 destruction is induced by UV radiation and TNF-α and coincides with a caspase-dependent apoptotic response. (A) MDA cells were irradiated (25 or 50 J/m 2 UV radiation) and returned to normal culture conditions in the presence or absence of 1 μM of the kinase inhibitor UCN-01 for 0–6 h. Proteins extracted at indicated times after irradiation were analyzed by immunoblotting. (B) MDA cells were treated with 1 μM UCN01, 40 nM adozelesin (Ado), or both (Ado+UCN01) for indicated times and extracted proteins analyzed by immunoblotting. (C) DNA was isolated from MDA cells treated with various combinations of Ado, 100 ng/ml TNF-α (TNF), and 10 μg/ml cycloheximide (TNF-CHX) with or without 10 μM MG132 (MG) or 10 μM caspase inhibitor I (CI) for indicated times and then size-fractionated on ethidium bromide-containing agarose gels. The standard (std) is a 100-base pair ladder. (D) Cells were treated with the indicated compounds for 0–6 h and protein extracts analyzed by immunoblotting.

    Techniques Used: Multiple Displacement Amplification, Irradiation, Isolation

    Adozelesin induces the proteasome-dependent destruction of Cdc6 in budding yeast. (A and C) S. cerevisiae cells harboring plasmids expressing the S. cerevisiae Cdc6 (scCdc6) or Arabidopsis CDC6 (AtCDC6) genes regulated by a galactose-inducible promoter were changed from raffinose- to galactose-containing medium to induce the ectopic expression of scCdc6 or AtCDC6. They were treated with 4 μM adozelesin and/or 250 μM MG132 beginning 1 h (scCdc6 experiments) or 1.5 h (AtCDC6 experiments) after galactose induction. Total protein lysates made at the indicated times after induction were analyzed by immunoblotting for S. cerevisiae Cdc6 (scCdc6) or arabidopsis Cdc6 (AtCDC6) proteins, or for scMcm2 protein as a control. (B) cdc4 cells were synchronized in early S phase by release from an α factor G1 block into HU and then cultured in the continued presence of HU at the nonpermissive temperature (37°C) for 2 h to destroy the activity of Cdc4p. Cycloheximide and adozelesin were then added for the times indicated before cells were lysed for Western blot analysis. Arp, actin-related protein.
    Figure Legend Snippet: Adozelesin induces the proteasome-dependent destruction of Cdc6 in budding yeast. (A and C) S. cerevisiae cells harboring plasmids expressing the S. cerevisiae Cdc6 (scCdc6) or Arabidopsis CDC6 (AtCDC6) genes regulated by a galactose-inducible promoter were changed from raffinose- to galactose-containing medium to induce the ectopic expression of scCdc6 or AtCDC6. They were treated with 4 μM adozelesin and/or 250 μM MG132 beginning 1 h (scCdc6 experiments) or 1.5 h (AtCDC6 experiments) after galactose induction. Total protein lysates made at the indicated times after induction were analyzed by immunoblotting for S. cerevisiae Cdc6 (scCdc6) or arabidopsis Cdc6 (AtCDC6) proteins, or for scMcm2 protein as a control. (B) cdc4 cells were synchronized in early S phase by release from an α factor G1 block into HU and then cultured in the continued presence of HU at the nonpermissive temperature (37°C) for 2 h to destroy the activity of Cdc4p. Cycloheximide and adozelesin were then added for the times indicated before cells were lysed for Western blot analysis. Arp, actin-related protein.

    Techniques Used: Expressing, Blocking Assay, Cell Culture, Activity Assay, Western Blot

    16) Product Images from "G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation"

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    Journal: bioRxiv

    doi: 10.1101/2021.02.05.430008

    MG132 does not prevent G9a/GLP-induced reduction of the ATG12-ATG5 conjugate. G9a/GLP expression decreased the ATG12-ATG5 conjugate level in Hela cells, and MG132 did not prevent the reduction. On the contrary, MG132 reduced the ATG12-ATG5 conjugate level through a lysosome-mediated protein degradation pathway based on the increased LC3BII level under growth ( a and c ), mTOR inhibition ( b ), and HEPES starvation ( d ) conditions. Therefore, G9a/GLP expression did not accumulate the ATG12-ATG5 conjugate level in the presence of MG132 (mean ± SEM of n = 3 replicates; *p
    Figure Legend Snippet: MG132 does not prevent G9a/GLP-induced reduction of the ATG12-ATG5 conjugate. G9a/GLP expression decreased the ATG12-ATG5 conjugate level in Hela cells, and MG132 did not prevent the reduction. On the contrary, MG132 reduced the ATG12-ATG5 conjugate level through a lysosome-mediated protein degradation pathway based on the increased LC3BII level under growth ( a and c ), mTOR inhibition ( b ), and HEPES starvation ( d ) conditions. Therefore, G9a/GLP expression did not accumulate the ATG12-ATG5 conjugate level in the presence of MG132 (mean ± SEM of n = 3 replicates; *p

    Techniques Used: Expressing, Inhibition

    Endogenous G9a undergoes degradation via the UPS during autophagy. Autophagy induction by serum starvation (HEPES) diminished the endogenous G9a protein level in Hela cells. In the presence of MG132, a proteasome inhibitor, endogenous G9a degradation was significantly reduced under starvation in HEPES medium, indicating that G9a degradation was facilitated by the UPS (mean ± SEM of n = 4 replicates; ***p
    Figure Legend Snippet: Endogenous G9a undergoes degradation via the UPS during autophagy. Autophagy induction by serum starvation (HEPES) diminished the endogenous G9a protein level in Hela cells. In the presence of MG132, a proteasome inhibitor, endogenous G9a degradation was significantly reduced under starvation in HEPES medium, indicating that G9a degradation was facilitated by the UPS (mean ± SEM of n = 4 replicates; ***p

    Techniques Used:

    Calpains cleave G9a protein for ubiquitin-mediated degradation. a, Immunoprecipitation (IP) assays show that G9a bound to calpain 1 and 2. 293T cells were co-transfected with 5xMyc-calpain and G9a-GFP plasmids, and samples were prepared as described in the STAR Methods. To prevent G9a cleavage by calpains, the calpain inhibitor calpeptin (10 μM), protease inhibitors, and EGTA (1 mM) were added. b, G9a was cleaved by elevated Ca 2+ influx (A23187) and the expression of calpains in 293T cells. The accumulation of cleaved G9a fragments by MG132 indicates that the cleaved G9a was degraded by the UPS. c, Expression of calpain 1 or calpain 2 promoted G9a ubiquitination in 293T cells. 293T cells were co-transfected with the indicated constructs. d, Elevated Ca 2+ influx by A23187 promoted G9a ubiquitination, whereas Ca 2+ chelation by BAPTA/AM reduced G9a ubiquitination under starvation in EBSS medium. GST pull-down assays were performed to monitor the ubiquitination level of G9a-GST after 12 hours of EBSS starvation. e, Calpain cleavage sites in G9a are located between aa 600 and aa 700. f, The Δ600–700 G9a-GFP mutant lacking the calpain cleavage sites was more resistant than WT G9a-GFP to calpain 1, as indicated by the lack of cleavage products (red arrow) and a higher level of intact G9a protein. The cleavage products of the Δ600–700 G9a (black arrow) suggest the presence of an additional calpain cleavage site at the N-terminus of G9a. g, An in vitro calpain cleavage assay confirmed that G9a has two cleavage sites. The main site is between aa 600 and aa 700. An additional N-terminal cleavage site is near aa 300. Recombinant Flag-tagged hG9a, calpain 1, and calpain S1 (a calpain 1 co-activator) were incubated for 10 min at 37°C, and the reaction products were subjected to Western blot analysis. h, A schematic diagram of the G9a cleavage sites and fragments in Fig. 2f , g and Supplementary Fig. 5 . The calpain cleavage sequences in G9a are highly conserved across vertebrates.
    Figure Legend Snippet: Calpains cleave G9a protein for ubiquitin-mediated degradation. a, Immunoprecipitation (IP) assays show that G9a bound to calpain 1 and 2. 293T cells were co-transfected with 5xMyc-calpain and G9a-GFP plasmids, and samples were prepared as described in the STAR Methods. To prevent G9a cleavage by calpains, the calpain inhibitor calpeptin (10 μM), protease inhibitors, and EGTA (1 mM) were added. b, G9a was cleaved by elevated Ca 2+ influx (A23187) and the expression of calpains in 293T cells. The accumulation of cleaved G9a fragments by MG132 indicates that the cleaved G9a was degraded by the UPS. c, Expression of calpain 1 or calpain 2 promoted G9a ubiquitination in 293T cells. 293T cells were co-transfected with the indicated constructs. d, Elevated Ca 2+ influx by A23187 promoted G9a ubiquitination, whereas Ca 2+ chelation by BAPTA/AM reduced G9a ubiquitination under starvation in EBSS medium. GST pull-down assays were performed to monitor the ubiquitination level of G9a-GST after 12 hours of EBSS starvation. e, Calpain cleavage sites in G9a are located between aa 600 and aa 700. f, The Δ600–700 G9a-GFP mutant lacking the calpain cleavage sites was more resistant than WT G9a-GFP to calpain 1, as indicated by the lack of cleavage products (red arrow) and a higher level of intact G9a protein. The cleavage products of the Δ600–700 G9a (black arrow) suggest the presence of an additional calpain cleavage site at the N-terminus of G9a. g, An in vitro calpain cleavage assay confirmed that G9a has two cleavage sites. The main site is between aa 600 and aa 700. An additional N-terminal cleavage site is near aa 300. Recombinant Flag-tagged hG9a, calpain 1, and calpain S1 (a calpain 1 co-activator) were incubated for 10 min at 37°C, and the reaction products were subjected to Western blot analysis. h, A schematic diagram of the G9a cleavage sites and fragments in Fig. 2f , g and Supplementary Fig. 5 . The calpain cleavage sequences in G9a are highly conserved across vertebrates.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Mutagenesis, In Vitro, Cleavage Assay, Recombinant, Incubation, Western Blot

    G9a methylates K72 residue in ATG12. a, GST pull-down assays show a direct interaction of G9a with ATG12, ATG5, and LC3B. b, IP assays reveal a specific interaction of endogenous G9a with ATG12 and ATG5 in 293T cells. c, Mass spectrometry analysis confirmed mono- and di-methylation at K72 residue in ATG12. The GST-ATG12-B used in Fig. 3d was subjected to Glu-C endoproteinase digestion and LC-MS/MS analysis. d, K72R ATG12 was not methylated by either endogenous or exogenous G9a/GLP, suggesting that G9a and GLP specifically methylate K72 in ATG12. WT ATG12 was more extensively mono-methylated by G9a/GLP than K71R ATG12 in 293T cells. e, K72 mono-methylation (K72me1) by endogenous G9a/GLP was reversed by JMJD2C expression in Hela cells. f, MG132 caused the accumulation of ATG12 K72me1 in WT ATG12 and K71R ATG12 in Hela cells, confirming that K72 methylation led to ATG12 degradation by the UPS pathway. g, Inhibition of G9a/GLP activity by UNC0638 reduced the ATG12 K72me1 level in Hela cells. h, MG132 increased the ATG12 K72me1 level in Hela cells under non-stress conditions. i, Leptomycin B decreased the ATG12 K72me1 level but increased the protein levels of the ATG12-ATG5 conjugate in the cytoplasm and G9a in the nucleus in Hela cells (mean ± SEM of n = 3 replicates; *p
    Figure Legend Snippet: G9a methylates K72 residue in ATG12. a, GST pull-down assays show a direct interaction of G9a with ATG12, ATG5, and LC3B. b, IP assays reveal a specific interaction of endogenous G9a with ATG12 and ATG5 in 293T cells. c, Mass spectrometry analysis confirmed mono- and di-methylation at K72 residue in ATG12. The GST-ATG12-B used in Fig. 3d was subjected to Glu-C endoproteinase digestion and LC-MS/MS analysis. d, K72R ATG12 was not methylated by either endogenous or exogenous G9a/GLP, suggesting that G9a and GLP specifically methylate K72 in ATG12. WT ATG12 was more extensively mono-methylated by G9a/GLP than K71R ATG12 in 293T cells. e, K72 mono-methylation (K72me1) by endogenous G9a/GLP was reversed by JMJD2C expression in Hela cells. f, MG132 caused the accumulation of ATG12 K72me1 in WT ATG12 and K71R ATG12 in Hela cells, confirming that K72 methylation led to ATG12 degradation by the UPS pathway. g, Inhibition of G9a/GLP activity by UNC0638 reduced the ATG12 K72me1 level in Hela cells. h, MG132 increased the ATG12 K72me1 level in Hela cells under non-stress conditions. i, Leptomycin B decreased the ATG12 K72me1 level but increased the protein levels of the ATG12-ATG5 conjugate in the cytoplasm and G9a in the nucleus in Hela cells (mean ± SEM of n = 3 replicates; *p

    Techniques Used: Mass Spectrometry, Methylation, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition, Activity Assay

    17) Product Images from "Bimodal regulation of ICR1 levels generates self-organizing auxin distribution"

    Article Title: Bimodal regulation of ICR1 levels generates self-organizing auxin distribution

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1413918111

    GFP-ICR1 decay during root regeneration requires polar auxin transport, auxin-induced gene expression, de novo protein synthesis, and proteasome activity. ( A ) NPA inhibited GFP-ICR1 decay. DII-VENUS, DR5 rev ::GFP, and GFP-ICR1 show the changes in auxin response and ICR1 stability in sectioned roots treated with the auxin transport inhibitor NPA. ( B ) DII-VENUS and GFP-ICR1 levels in sectioned root tips in the control and after incubation in the SCF (TIR1/AFB) inhibitor auxinole, the protein translation inhibitor CHX, and the proteasome inhibitor MG132. Arrowheads highlight the sites of auxin accumulation and GFP-ICR1 decay. (Scale bars: 50 μm.)
    Figure Legend Snippet: GFP-ICR1 decay during root regeneration requires polar auxin transport, auxin-induced gene expression, de novo protein synthesis, and proteasome activity. ( A ) NPA inhibited GFP-ICR1 decay. DII-VENUS, DR5 rev ::GFP, and GFP-ICR1 show the changes in auxin response and ICR1 stability in sectioned roots treated with the auxin transport inhibitor NPA. ( B ) DII-VENUS and GFP-ICR1 levels in sectioned root tips in the control and after incubation in the SCF (TIR1/AFB) inhibitor auxinole, the protein translation inhibitor CHX, and the proteasome inhibitor MG132. Arrowheads highlight the sites of auxin accumulation and GFP-ICR1 decay. (Scale bars: 50 μm.)

    Techniques Used: Expressing, Activity Assay, Incubation, Ziehl-Neelsen Stain

    18) Product Images from "Characterization of kinases involved in the phosphorylation of aggregated α-synuclein"

    Article Title: Characterization of kinases involved in the phosphorylation of aggregated α-synuclein

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.22537

    Examination of kinases that affect the phosphorylation of aggregated α-syn by treating cells with inhibitors after cellular seeding of α-syn. QBI293 cells were transfected with a plasmid for the expression of α-syn and then treated with recombinant 21-140 α-syn fibrils. The following drug treatments were initiated at 8 hours of calcium phosphate removal, as indicated in “Materials and Methods”: DMAT (25 μM) or TBCA (20 μM) for CK2 inhibition, BI2536 (1 μM) or GW843682X (GWX; 1 μM) for PLK inhibition, D4476 (10 μM) for CK1 inhibition, and MG132 (10 μM) for proteasome inhibition. Cells were harvested 16 hours after treatment. (A) Representative immunoblots of biochemical cellular fractionation of QBI293, separating triton X-100 (TX)-soluble α-syn protein from TX-insoluble protein. Phosphorylation of α-syn was assessed using antibody pSer129, and total α-syn was assessed with SNL4. (B) Quantitative analyses of the amount of pSer129 immunoreactivity in the TX-soluble fraction. Analysis was performed using a ratio of pSer129 to SNL4 immunoreactivity, and calculated as a percent of that observed in the DMSO control condition. Quantitation of the MG132 condition is indicated with the y-axis values at the far right. CK2 and PLK inhibitors significantly decreased, and MG132 significantly increased relative phosphorylation. p = 0.0006 by Kruskal-Wallis non-parametric ANOVA. Post-tests were performed by one-sample t-test since all conditions were standardized to DMSO control (*, p=0.04; **, p=
    Figure Legend Snippet: Examination of kinases that affect the phosphorylation of aggregated α-syn by treating cells with inhibitors after cellular seeding of α-syn. QBI293 cells were transfected with a plasmid for the expression of α-syn and then treated with recombinant 21-140 α-syn fibrils. The following drug treatments were initiated at 8 hours of calcium phosphate removal, as indicated in “Materials and Methods”: DMAT (25 μM) or TBCA (20 μM) for CK2 inhibition, BI2536 (1 μM) or GW843682X (GWX; 1 μM) for PLK inhibition, D4476 (10 μM) for CK1 inhibition, and MG132 (10 μM) for proteasome inhibition. Cells were harvested 16 hours after treatment. (A) Representative immunoblots of biochemical cellular fractionation of QBI293, separating triton X-100 (TX)-soluble α-syn protein from TX-insoluble protein. Phosphorylation of α-syn was assessed using antibody pSer129, and total α-syn was assessed with SNL4. (B) Quantitative analyses of the amount of pSer129 immunoreactivity in the TX-soluble fraction. Analysis was performed using a ratio of pSer129 to SNL4 immunoreactivity, and calculated as a percent of that observed in the DMSO control condition. Quantitation of the MG132 condition is indicated with the y-axis values at the far right. CK2 and PLK inhibitors significantly decreased, and MG132 significantly increased relative phosphorylation. p = 0.0006 by Kruskal-Wallis non-parametric ANOVA. Post-tests were performed by one-sample t-test since all conditions were standardized to DMSO control (*, p=0.04; **, p=

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Recombinant, Inhibition, Western Blot, Cell Fractionation, Quantitation Assay

    19) Product Images from "Regulation of cytokine-inducible SH2-containing protein (CIS) by ubiquitination and Elongin B/C interaction"

    Article Title: Regulation of cytokine-inducible SH2-containing protein (CIS) by ubiquitination and Elongin B/C interaction

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2014.10.017

    CIS is degraded and interacts with ubiquitin. A ,PRLR-HA CAD stable cells were treated with PRL and DMSO (−) or MG132 (+)for 18 hrs. Lysates (50 µg) were analyzed by Western blot using anti-CISand anti-tubulin antibodies. An increased
    Figure Legend Snippet: CIS is degraded and interacts with ubiquitin. A ,PRLR-HA CAD stable cells were treated with PRL and DMSO (−) or MG132 (+)for 18 hrs. Lysates (50 µg) were analyzed by Western blot using anti-CISand anti-tubulin antibodies. An increased

    Techniques Used: Western Blot

    CIS is ubiquitinated. A and B , HEK 293t cells weretransfected with combinations of the indicated FLAG-ubiquitin with HA-CIS,HA-6KR or HA-SOCS3 expression constructs, as indicated in the matrix above A , and treated with MG132. Cell lysates wereimmunoprecipitated
    Figure Legend Snippet: CIS is ubiquitinated. A and B , HEK 293t cells weretransfected with combinations of the indicated FLAG-ubiquitin with HA-CIS,HA-6KR or HA-SOCS3 expression constructs, as indicated in the matrix above A , and treated with MG132. Cell lysates wereimmunoprecipitated

    Techniques Used: Expressing, Construct

    20) Product Images from "Proteasome-dependent degradation of the human estrogen receptor"

    Article Title: Proteasome-dependent degradation of the human estrogen receptor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    The proteasome inhibitors, MG132 and lactacystin, block ER degradation in vitro . ( A ) 35 S-labeled ER protein was synthesized in vitro in the presence of either vehicle only, 33 μM MG132 or 33 μM lactacystin with TNT-coupled rabbit reticulocyte extracts. The labeled ER protein was then incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs (UbcH5B and UbcH7). Arrows indicate the position of intact and degraded ER protein. ( B ) The UBA/UBCs and proteasome inhibitors, MG132 and lactacystin, promote the accumulation of slower migrating forms of ER (shown by a bracket). 35 S-labeled ER protein was synthesized in vitro in the presence of either vehicle only, 33 μM MG132 or 33 μM lactacystin with TNT-coupled rabbit reticulocyte extracts. The labeled ER protein was then incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs (UbcH5B and UbcH7). Then the ER protein was analyzed by Western blot analysis using H222 antibody that specifically recognizes ER. The control lane contains reticulocyte extract only. Arrows indicate the position of intact, degraded, and slower migrating forms of ER protein.
    Figure Legend Snippet: The proteasome inhibitors, MG132 and lactacystin, block ER degradation in vitro . ( A ) 35 S-labeled ER protein was synthesized in vitro in the presence of either vehicle only, 33 μM MG132 or 33 μM lactacystin with TNT-coupled rabbit reticulocyte extracts. The labeled ER protein was then incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs (UbcH5B and UbcH7). Arrows indicate the position of intact and degraded ER protein. ( B ) The UBA/UBCs and proteasome inhibitors, MG132 and lactacystin, promote the accumulation of slower migrating forms of ER (shown by a bracket). 35 S-labeled ER protein was synthesized in vitro in the presence of either vehicle only, 33 μM MG132 or 33 μM lactacystin with TNT-coupled rabbit reticulocyte extracts. The labeled ER protein was then incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs (UbcH5B and UbcH7). Then the ER protein was analyzed by Western blot analysis using H222 antibody that specifically recognizes ER. The control lane contains reticulocyte extract only. Arrows indicate the position of intact, degraded, and slower migrating forms of ER protein.

    Techniques Used: Blocking Assay, In Vitro, Labeling, Synthesized, Incubation, Western Blot

    PR and TR proteins are not the target of the ubiquitin–proteasome pathway. ( A ) 35 S-labeled PR protein was incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs for 120 min (UbcH5B and UbcH7). In the presence of UBA/UBCs, the reaction mixtures were treated with either vehicle or 33 μM MG132. The position of intact PR is indicated by the arrow. ( B ) 35 S-labeled TR was incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs for 120 min (UbcH5B and UbcH7). The reaction mixtures containing UBA and UBCs were treated with either vehicle or 33 μM MG132. The position of intact TR is indicated by the arrow.
    Figure Legend Snippet: PR and TR proteins are not the target of the ubiquitin–proteasome pathway. ( A ) 35 S-labeled PR protein was incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs for 120 min (UbcH5B and UbcH7). In the presence of UBA/UBCs, the reaction mixtures were treated with either vehicle or 33 μM MG132. The position of intact PR is indicated by the arrow. ( B ) 35 S-labeled TR was incubated with ATP and ubiquitin either in the absence of UBA/UBCs or in the presence of UBA and UBCs for 120 min (UbcH5B and UbcH7). The reaction mixtures containing UBA and UBCs were treated with either vehicle or 33 μM MG132. The position of intact TR is indicated by the arrow.

    Techniques Used: Labeling, Incubation

    21) Product Images from "P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase complex HSP70/CHIP"

    Article Title: P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase complex HSP70/CHIP

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.79

    The p42 promotes p85 degradation through ubiquitin–proteasome system. ( a ) GFP-p42 (1 and 3 μ g) was transfected into HEK293 cells, following exposure to 10 μ M MG132 for 8 h. Immunoblotting was conducted to monitor p85, p42 or β -actin levels. ( b ) PC12 cells were co-transfected with HA-Ub and Flag-p85 and/or GFP-p42/p48 for 30 h and treated with MG132 (10 μ M) for an additional 8 h. To obtain similar amount of immunoprecipitated Flag-p85, we transfected 0.5 μ g of GFP-p42 while we transfected 1 μ g of GFP or GFP-p48. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA antibody. ( c ) HEK 293T cells were co-transfected with Flag-p85 and HA-Ub along with N-si-p48 or si-Ebp1 following exposure of MG132. Ubiquitinated p85 was detected by immunoblotting with anti-HA antibody. ( d ) Cells were transfected with various Flag-p85 fragments with or without GFP-p42, and p85 levels were determined by anti-Flag antibody (left). Densitometry analysis (right). ** P
    Figure Legend Snippet: The p42 promotes p85 degradation through ubiquitin–proteasome system. ( a ) GFP-p42 (1 and 3 μ g) was transfected into HEK293 cells, following exposure to 10 μ M MG132 for 8 h. Immunoblotting was conducted to monitor p85, p42 or β -actin levels. ( b ) PC12 cells were co-transfected with HA-Ub and Flag-p85 and/or GFP-p42/p48 for 30 h and treated with MG132 (10 μ M) for an additional 8 h. To obtain similar amount of immunoprecipitated Flag-p85, we transfected 0.5 μ g of GFP-p42 while we transfected 1 μ g of GFP or GFP-p48. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA antibody. ( c ) HEK 293T cells were co-transfected with Flag-p85 and HA-Ub along with N-si-p48 or si-Ebp1 following exposure of MG132. Ubiquitinated p85 was detected by immunoblotting with anti-HA antibody. ( d ) Cells were transfected with various Flag-p85 fragments with or without GFP-p42, and p85 levels were determined by anti-Flag antibody (left). Densitometry analysis (right). ** P

    Techniques Used: Transfection, Immunoprecipitation

    22) Product Images from "SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1"

    Article Title: SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00426-16

    ICP0 disperses PIAS1 away from domains that contain infecting HSV-1 genomes without targeting it for proteasomal degradation. (A) Western blots show PIAS1 protein levels during wild-type or ICP0-null mutant HSV-1 infection. HFt cells were infected with 10 PFU of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132. Whole-cell lysates were harvested at 3, 6, or 9 h postinfection (hpi), and proteins were resolved by Tris-Tricine SDS-PAGE. Membranes were probed for PIAS1, PML as an example of an ICP0 substrate, ICP0, ICP4, and UL42 to show the progression of infection, and actin as a loading control. Molecular masses are indicated. (B) Bar graph shows the average relative levels of PIAS1 during infection with wild-type or ICP0-null mutant HSV-1. The intensities of PIAS1 protein bands were quantitated from Western blots as in panel A, normalized to their respective loading control, and presented as a ratio to the level in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown ( n = 7). *, P
    Figure Legend Snippet: ICP0 disperses PIAS1 away from domains that contain infecting HSV-1 genomes without targeting it for proteasomal degradation. (A) Western blots show PIAS1 protein levels during wild-type or ICP0-null mutant HSV-1 infection. HFt cells were infected with 10 PFU of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132. Whole-cell lysates were harvested at 3, 6, or 9 h postinfection (hpi), and proteins were resolved by Tris-Tricine SDS-PAGE. Membranes were probed for PIAS1, PML as an example of an ICP0 substrate, ICP0, ICP4, and UL42 to show the progression of infection, and actin as a loading control. Molecular masses are indicated. (B) Bar graph shows the average relative levels of PIAS1 during infection with wild-type or ICP0-null mutant HSV-1. The intensities of PIAS1 protein bands were quantitated from Western blots as in panel A, normalized to their respective loading control, and presented as a ratio to the level in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown ( n = 7). *, P

    Techniques Used: Western Blot, Mutagenesis, Infection, SDS Page

    23) Product Images from "Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity"

    Article Title: Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

    Journal: Autophagy

    doi: 10.4161/15548627.2014.981914

    Proteasomal degradation of free ATG12 protein occurs independent of autophagy ( A ) E1A and Ras -transformed WT or Atg7 knockout MEF were treated for 8 h with MG132 and cell lysates were probed for ATG12 expression. ( B ) Atg3 or ( C ) Atg5 knockout MEFs were treated with MG132 for 4 h and 8 h and analyzed for ATG12 expression. ( D ) U2OS cells expressing ATG12 G140A were treated for 8 h with MG132 and/or CHX as indicated and lysates were examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.
    Figure Legend Snippet: Proteasomal degradation of free ATG12 protein occurs independent of autophagy ( A ) E1A and Ras -transformed WT or Atg7 knockout MEF were treated for 8 h with MG132 and cell lysates were probed for ATG12 expression. ( B ) Atg3 or ( C ) Atg5 knockout MEFs were treated with MG132 for 4 h and 8 h and analyzed for ATG12 expression. ( D ) U2OS cells expressing ATG12 G140A were treated for 8 h with MG132 and/or CHX as indicated and lysates were examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.

    Techniques Used: Transformation Assay, Knock-Out, Expressing, Western Blot, Activated Clotting Time Assay

    Free ATG12 is rapidly degraded in a proteasome-dependent manner ( A ) Western blot detection of ATG12 in U2OS cells. ( B ) Endogenous ATG12 expression in U2OS cells following CHX treatment. ( C ) U2OS expressing empty vector or ATG12 were treated with CHX for various times and probed for ATG12 expression. ( D ) U2OS cells were treated were treated for 8 h as indicated with MG132 and/or CHX and probed for ATG12 expression. ( E ) U2OS were treated for 8 h with MG132 or lactacystin and examined for ATG12 expression. ( F ) U2OS expressing ATG12 were treated for 8 h with MG132 and/or CHX as indicated and cell lysates were probed for ATG12 expression. ACT or TOMM20 were used as a loading control.
    Figure Legend Snippet: Free ATG12 is rapidly degraded in a proteasome-dependent manner ( A ) Western blot detection of ATG12 in U2OS cells. ( B ) Endogenous ATG12 expression in U2OS cells following CHX treatment. ( C ) U2OS expressing empty vector or ATG12 were treated with CHX for various times and probed for ATG12 expression. ( D ) U2OS cells were treated were treated for 8 h as indicated with MG132 and/or CHX and probed for ATG12 expression. ( E ) U2OS were treated for 8 h with MG132 or lactacystin and examined for ATG12 expression. ( F ) U2OS expressing ATG12 were treated for 8 h with MG132 and/or CHX as indicated and cell lysates were probed for ATG12 expression. ACT or TOMM20 were used as a loading control.

    Techniques Used: Western Blot, Expressing, Plasmid Preparation, Activated Clotting Time Assay

    Free ATG12 promotes proteasome inhibitor-mediated cell death ( A ) U2OS cells stably expressing empty vector or BCL2L1 were treated with MG132 and cell viability was determined by SYTOX Green staining using an Incucyte Imager; data represents the mean +/− standard error of the mean (SEM) of 3 experiments at a representative time-point (24 h). ( B ) U2OS cells were assessed for ATG12 expression 2 d post-transfection with control or ATG12 siRNA. Cell viability of control or ATG12 siRNA-transfected U2OS cells, treated with MG132 ( C ) HBSS ( D ) or Act D ( E ) was determined by SYTOX Green staining using an Incucyte Imager; representative time-points shown (24 h MG132, 30 h HBSS, 24 h Act D). Graphs represent the mean +/− SEM of 4 experiments. ( F ) U2OS, transfected with control or ATG12 siRNA, or stably expressing vector or BCL2L1 were starved in HBSS. After 48 h of starvation, cells were washed, cultivated for 7 d in DMEM and colonies were stained with methylene blue. ( G ) U2OS cells were treated with HBSS (24 h), MG132 (16 h) or Act D (16 h) and examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.
    Figure Legend Snippet: Free ATG12 promotes proteasome inhibitor-mediated cell death ( A ) U2OS cells stably expressing empty vector or BCL2L1 were treated with MG132 and cell viability was determined by SYTOX Green staining using an Incucyte Imager; data represents the mean +/− standard error of the mean (SEM) of 3 experiments at a representative time-point (24 h). ( B ) U2OS cells were assessed for ATG12 expression 2 d post-transfection with control or ATG12 siRNA. Cell viability of control or ATG12 siRNA-transfected U2OS cells, treated with MG132 ( C ) HBSS ( D ) or Act D ( E ) was determined by SYTOX Green staining using an Incucyte Imager; representative time-points shown (24 h MG132, 30 h HBSS, 24 h Act D). Graphs represent the mean +/− SEM of 4 experiments. ( F ) U2OS, transfected with control or ATG12 siRNA, or stably expressing vector or BCL2L1 were starved in HBSS. After 48 h of starvation, cells were washed, cultivated for 7 d in DMEM and colonies were stained with methylene blue. ( G ) U2OS cells were treated with HBSS (24 h), MG132 (16 h) or Act D (16 h) and examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Staining, Transfection, Activated Clotting Time Assay, Western Blot

    Direct ubiquitination of free ATG12 regulates its proteasomal degradation ( A ) 293T cells expressing vector or ATG12 and His-ubiquitin were treated with MG132 as indicated. Cell lysates were subject to His-tag affinity isolation and immunoblotted for ATG12 and ubiquitin expression ( B ) Atg12 knockout MEFs, as well as MEFs stably expressing Atg12 , were treated for 4 h with chloroquine, then examined for ATG12 and LC3B expression. ( C ) 293T cells expressing ATG12 or ATG12[K-] together with His-ubiquitin, were treated as indicated, subject to His-tag affinity isolation and probed for ATG12 and ubiquitin expression. ( D ) U2OS cells expressing ATG12 or ATG12[K-] and treated for 6 h with CHX and analyzed for ATG12 and TOMM20 expression, densitometric analysis was performed using ImageJ software, normalizing to TOMM20 levels. ( E ) U2OS cells expressing ATG12[K-] were treated with MG132 and/or CHX for 8 h as indicated and examined for ATG12 expression. ACT or TOMM20 were used as loading controls.
    Figure Legend Snippet: Direct ubiquitination of free ATG12 regulates its proteasomal degradation ( A ) 293T cells expressing vector or ATG12 and His-ubiquitin were treated with MG132 as indicated. Cell lysates were subject to His-tag affinity isolation and immunoblotted for ATG12 and ubiquitin expression ( B ) Atg12 knockout MEFs, as well as MEFs stably expressing Atg12 , were treated for 4 h with chloroquine, then examined for ATG12 and LC3B expression. ( C ) 293T cells expressing ATG12 or ATG12[K-] together with His-ubiquitin, were treated as indicated, subject to His-tag affinity isolation and probed for ATG12 and ubiquitin expression. ( D ) U2OS cells expressing ATG12 or ATG12[K-] and treated for 6 h with CHX and analyzed for ATG12 and TOMM20 expression, densitometric analysis was performed using ImageJ software, normalizing to TOMM20 levels. ( E ) U2OS cells expressing ATG12[K-] were treated with MG132 and/or CHX for 8 h as indicated and examined for ATG12 expression. ACT or TOMM20 were used as loading controls.

    Techniques Used: Expressing, Plasmid Preparation, Isolation, Knock-Out, Stable Transfection, Software, Activated Clotting Time Assay

    Free ATG12 promotes cell death independent of autophagy ( A ) U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4B C74A were treated for 4 h with chloroquine and cell lysates were blotted for RFP and LC3B. U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4B C74A were transfected with control or ATG12 siRNA. ( B ) Following MG132 treatment (24 h) or ( C ) HBSS starvation (48 h), cell viability was determined by SYTOX Green exclusion in an Incuyte Imager. Graphs show the mean +/− SEM of 3 ( B ) or 5 ( C ) experiments at representative time-points (24h MG132, 48 h HBSS). Western blots show cell lysates of U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A, transfected with control or ATG12 siRNA, treated for 8 h with MG132 ( B ) or HBSS ( C ) and probed for RFP, LC3B, and ATG12. In all immunoblots, ACT or TOMM20 were used as a loading control.
    Figure Legend Snippet: Free ATG12 promotes cell death independent of autophagy ( A ) U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4B C74A were treated for 4 h with chloroquine and cell lysates were blotted for RFP and LC3B. U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4B C74A were transfected with control or ATG12 siRNA. ( B ) Following MG132 treatment (24 h) or ( C ) HBSS starvation (48 h), cell viability was determined by SYTOX Green exclusion in an Incuyte Imager. Graphs show the mean +/− SEM of 3 ( B ) or 5 ( C ) experiments at representative time-points (24h MG132, 48 h HBSS). Western blots show cell lysates of U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A, transfected with control or ATG12 siRNA, treated for 8 h with MG132 ( B ) or HBSS ( C ) and probed for RFP, LC3B, and ATG12. In all immunoblots, ACT or TOMM20 were used as a loading control.

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Western Blot, Activated Clotting Time Assay

    24) Product Images from "Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy *"

    Article Title: Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.642900

    Syk associates with SGs in multiple cell types. A , MCF7-BD cells stably expressing Syk-EGFP were treated with sodium arsenite for 2 h or either MG132 or DMSO (solvent carrier for MG132) for 3 h. Cells were fixed, stained using antibodies against G3BP
    Figure Legend Snippet: Syk associates with SGs in multiple cell types. A , MCF7-BD cells stably expressing Syk-EGFP were treated with sodium arsenite for 2 h or either MG132 or DMSO (solvent carrier for MG132) for 3 h. Cells were fixed, stained using antibodies against G3BP

    Techniques Used: Stable Transfection, Expressing, Staining

    Syk catalyzes the phosphorylation of proteins on tyrosine within SGs. A , MCF7-BD cells lacking Syk (− Syk ) or expressing Syk-EGFP were treated with MG132 for 3 h, fixed, stained with antibodies against TIAR ( blue ) or phosphotyrosine ( pTyr ) ( red
    Figure Legend Snippet: Syk catalyzes the phosphorylation of proteins on tyrosine within SGs. A , MCF7-BD cells lacking Syk (− Syk ) or expressing Syk-EGFP were treated with MG132 for 3 h, fixed, stained with antibodies against TIAR ( blue ) or phosphotyrosine ( pTyr ) ( red

    Techniques Used: Expressing, Staining

    Syk promotes the clearance of SGs. A , MCF7-BD cells lacking Syk (− Syk ) or expressing Syk-EGFP (+ Syk ) were treated with MG132 for 3 h. Cells were fixed and stained with antibodies against G3BP. The number of SGs present per cell was counted. B
    Figure Legend Snippet: Syk promotes the clearance of SGs. A , MCF7-BD cells lacking Syk (− Syk ) or expressing Syk-EGFP (+ Syk ) were treated with MG132 for 3 h. Cells were fixed and stained with antibodies against G3BP. The number of SGs present per cell was counted. B

    Techniques Used: Expressing, Staining

    The phosphorylation of Syk on tyrosines 342 and 346 promotes its binding to SGs. MCF7-BD cells stably expressing Syk-EGFP ( WT ), Syk-EGFP(K396R) ( KD ), Syk-EGFP(Y342F/Y346F) ( 2F ), Syk-EGFP(Y342F) ( 342F ), or Syk-EGFP(Y346F) ( 346F ) were treated with MG132
    Figure Legend Snippet: The phosphorylation of Syk on tyrosines 342 and 346 promotes its binding to SGs. MCF7-BD cells stably expressing Syk-EGFP ( WT ), Syk-EGFP(K396R) ( KD ), Syk-EGFP(Y342F/Y346F) ( 2F ), Syk-EGFP(Y342F) ( 342F ), or Syk-EGFP(Y346F) ( 346F ) were treated with MG132

    Techniques Used: Binding Assay, Stable Transfection, Expressing

    Syk promotes the clearance of SGs through autophagy. A , MCF7-BD cells expressing Syk-EGFP were treated with MG132 for 3 h and then washed and placed in fresh medium lacking MG132 but containing DeBQ, 5-MA, or DMSO carrier for 4 h. Cells were fixed and
    Figure Legend Snippet: Syk promotes the clearance of SGs through autophagy. A , MCF7-BD cells expressing Syk-EGFP were treated with MG132 for 3 h and then washed and placed in fresh medium lacking MG132 but containing DeBQ, 5-MA, or DMSO carrier for 4 h. Cells were fixed and

    Techniques Used: Expressing

    Syk associates with SG markers. A , MCF7-BD cells expressing Syk-EGFP were treated with MG132 for the indicated times. Lysates were separated into detergent-soluble and -insoluble fractions, which were examined by Western blotting ( WB ) for Syk-EGFP ( Syk
    Figure Legend Snippet: Syk associates with SG markers. A , MCF7-BD cells expressing Syk-EGFP were treated with MG132 for the indicated times. Lysates were separated into detergent-soluble and -insoluble fractions, which were examined by Western blotting ( WB ) for Syk-EGFP ( Syk

    Techniques Used: Expressing, Western Blot

    SG clearance requires Syk activity and its association with SGs. A , MCF7-BD cells lacking Syk or expressing either Syk-EGFP ( Syk ) or Syk-AQL-EGFP ( Syk-AQL ) were treated with MG132 for 3 h and then washed and placed in fresh medium for 4 h. Cells were
    Figure Legend Snippet: SG clearance requires Syk activity and its association with SGs. A , MCF7-BD cells lacking Syk or expressing either Syk-EGFP ( Syk ) or Syk-AQL-EGFP ( Syk-AQL ) were treated with MG132 for 3 h and then washed and placed in fresh medium for 4 h. Cells were

    Techniques Used: Activity Assay, Expressing

    Grb7 promotes the binding of Syk to SGs. A , MCF7-BD cells expressing Syk-EGFP were treated with MG132 for 3 h, fixed, stained with antibodies against endogenous TIAR ( blue ) and Grb7 ( red ), and examined by confocal microscopy. B and C , MCF7-BD cells expressing
    Figure Legend Snippet: Grb7 promotes the binding of Syk to SGs. A , MCF7-BD cells expressing Syk-EGFP were treated with MG132 for 3 h, fixed, stained with antibodies against endogenous TIAR ( blue ) and Grb7 ( red ), and examined by confocal microscopy. B and C , MCF7-BD cells expressing

    Techniques Used: Binding Assay, Expressing, Staining, Confocal Microscopy

    Syk interacts with Grb7. A , MCF7-BD cells stably expressing Syk-EGFP were treated with MG132 in the presence of PP1 or DMSO carrier for 3 h, fixed, and stained for G3BP. Examples of SGs are indicated by the arrows in the illustrated merged images. Bar
    Figure Legend Snippet: Syk interacts with Grb7. A , MCF7-BD cells stably expressing Syk-EGFP were treated with MG132 in the presence of PP1 or DMSO carrier for 3 h, fixed, and stained for G3BP. Examples of SGs are indicated by the arrows in the illustrated merged images. Bar

    Techniques Used: Stable Transfection, Expressing, Staining

    25) Product Images from "The Human Spindle Assembly Checkpoint Protein Bub3 Is Required for the Establishment of Efficient Kinetochore-Microtubule Attachments"

    Article Title: The Human Spindle Assembly Checkpoint Protein Bub3 Is Required for the Establishment of Efficient Kinetochore-Microtubule Attachments

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-07-0633

    Misalignment defects after Bub3-depletion in comparison to those after Bub1 and BubR1 depletions. Immunofluorescence images of control and RNAi-depleted cells treated with MG132 and stained for BubR1 or Bub1 (green), Hec1 (red), tubulin (cyan), and DNA
    Figure Legend Snippet: Misalignment defects after Bub3-depletion in comparison to those after Bub1 and BubR1 depletions. Immunofluorescence images of control and RNAi-depleted cells treated with MG132 and stained for BubR1 or Bub1 (green), Hec1 (red), tubulin (cyan), and DNA

    Techniques Used: Immunofluorescence, Staining

    Misaligned chromosomes in Bub3-depleted cells often exhibit side-on attachments to microtubule walls. (A) Control, CENP-E, BubR1, Bub1, and Bub3 siRNA-depleted cells were incubated with MG132 and stained for CREST (red), tubulin (green), and DNA (blue).
    Figure Legend Snippet: Misaligned chromosomes in Bub3-depleted cells often exhibit side-on attachments to microtubule walls. (A) Control, CENP-E, BubR1, Bub1, and Bub3 siRNA-depleted cells were incubated with MG132 and stained for CREST (red), tubulin (green), and DNA (blue).

    Techniques Used: Incubation, Staining

    26) Product Images from "Functional analyses of a human vascular tumor FOS variant identify a novel degradation mechanism and a link to tumorigenesis"

    Article Title: Functional analyses of a human vascular tumor FOS variant identify a novel degradation mechanism and a link to tumorigenesis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.C117.815845

    A, FOS stability assay on HUVECs stably expressing FOS or FOSΔ. B, ubiquitin assay of cells transfected with the indicated constructs together with 10× HIS epitope-tagged ubiquitin. C, left panel, FOS stability assay on HUVECs stably expressing FOS in the presence or absence of MLN7243. Right panel, ubiquitin assay of cells transfected with the indicated constructs and cultured in the presence or absence of MG132 and MLN7243. IP, immunoprecipitation. D, in vitro translated FOS proteins were incubated with purified 20S proteasomes for the shown time course (minutes). 20S protein levels were determined by Western blotting using an antibody directed against PSMA1. 20S proteasome activity was independently quantified using the suc-Leu-Leu-Val-Tyr-AMC peptide (as shown in E ). E, experiment performed as in D .
    Figure Legend Snippet: A, FOS stability assay on HUVECs stably expressing FOS or FOSΔ. B, ubiquitin assay of cells transfected with the indicated constructs together with 10× HIS epitope-tagged ubiquitin. C, left panel, FOS stability assay on HUVECs stably expressing FOS in the presence or absence of MLN7243. Right panel, ubiquitin assay of cells transfected with the indicated constructs and cultured in the presence or absence of MG132 and MLN7243. IP, immunoprecipitation. D, in vitro translated FOS proteins were incubated with purified 20S proteasomes for the shown time course (minutes). 20S protein levels were determined by Western blotting using an antibody directed against PSMA1. 20S proteasome activity was independently quantified using the suc-Leu-Leu-Val-Tyr-AMC peptide (as shown in E ). E, experiment performed as in D .

    Techniques Used: Stability Assay, Stable Transfection, Expressing, Ubiquitin Assay, Transfection, Construct, Cell Culture, Immunoprecipitation, In Vitro, Incubation, Purification, Western Blot, Activity Assay

    27) Product Images from "Cinobufotalin powerfully reversed EBV-miR-BART22-induced cisplatin resistance via stimulating MAP2K4 to antagonize non-muscle myosin heavy chain IIA/glycogen synthase 3β/β-catenin signaling pathway"

    Article Title: Cinobufotalin powerfully reversed EBV-miR-BART22-induced cisplatin resistance via stimulating MAP2K4 to antagonize non-muscle myosin heavy chain IIA/glycogen synthase 3β/β-catenin signaling pathway

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.08.040

    MYH9 interacts with GSK3β to promote GSK3β ubiquitin protein degradation. (a) Immunofluorescence co-staining of GSK3β and MYH9 to detect colocalization. The fluorescence intensities along the red arrow crossing the cytoplasm were calculated to show the colocalisation of GSK3β and MYH9 , scale bar, 25 μm; Co-immunoprecipitation analysis of the effect of MYH9 on the interaction with GSK3β in HONE1-EBV + cells. (b) Western blotting analysis of the effect of MYH9 overexpression on GSK3β stability in NPC cells treated with cycloheximide and MG132 at different time points, GAPDH served as controls. (c) Co-immunoprecipitation analysis of the effect of MYH9 on the interaction between GSK3β, ubiquitin and TRAF6 in HONE1-EBV + cells. (d) Co-immunoprecipitation analysis of ubiquitin, TRAF6 and wild GSK3β or mutant GSK3β in MYH9-overexpressed HONE1-EBV + cells. (e) Immunofluorescence costaining of MYH9 and GSK3β expression and localization in MYH9 -overexpressed or MYH9 -depleted HONE1-EBV + and 5-8F cells. Scale bar, 25 μm. (f) Nucleic and cytoplasmic proteins were extracted for β-catenin detection by western blotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: MYH9 interacts with GSK3β to promote GSK3β ubiquitin protein degradation. (a) Immunofluorescence co-staining of GSK3β and MYH9 to detect colocalization. The fluorescence intensities along the red arrow crossing the cytoplasm were calculated to show the colocalisation of GSK3β and MYH9 , scale bar, 25 μm; Co-immunoprecipitation analysis of the effect of MYH9 on the interaction with GSK3β in HONE1-EBV + cells. (b) Western blotting analysis of the effect of MYH9 overexpression on GSK3β stability in NPC cells treated with cycloheximide and MG132 at different time points, GAPDH served as controls. (c) Co-immunoprecipitation analysis of the effect of MYH9 on the interaction between GSK3β, ubiquitin and TRAF6 in HONE1-EBV + cells. (d) Co-immunoprecipitation analysis of ubiquitin, TRAF6 and wild GSK3β or mutant GSK3β in MYH9-overexpressed HONE1-EBV + cells. (e) Immunofluorescence costaining of MYH9 and GSK3β expression and localization in MYH9 -overexpressed or MYH9 -depleted HONE1-EBV + and 5-8F cells. Scale bar, 25 μm. (f) Nucleic and cytoplasmic proteins were extracted for β-catenin detection by western blotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Immunofluorescence, Staining, Fluorescence, Immunoprecipitation, Western Blot, Over Expression, Mutagenesis, Expressing

    EBV-miR-BART22 activates the PI3K/AKT/GSK3β/β-catenin signaling pathway (a) Protein levels of PI3K, p-PI3K, AKT, p-AKT, c-Jun, β-catenin, GSK3β, p-GSK3β, c-Myc, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4 and Sox2 were measured by western blot after miR-BART22 overexpression. Levels of related proteins were reduced or increased after BART22 inhibitor transfection or LY294002 treatment. GAPDH served as controls. (b) Immunohistochemistry analysis of E-cadherin, N-cadherin, Nanog and OCT4 expression. Original magnification, ×200, scale bar, 100 μm. (c) Western blotting analysis of the effect of miR-BART22 overexpression on GSK3β stability in NPC cells treated with cycloheximide and MG132 at different time points, GAPDH served as controls.
    Figure Legend Snippet: EBV-miR-BART22 activates the PI3K/AKT/GSK3β/β-catenin signaling pathway (a) Protein levels of PI3K, p-PI3K, AKT, p-AKT, c-Jun, β-catenin, GSK3β, p-GSK3β, c-Myc, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4 and Sox2 were measured by western blot after miR-BART22 overexpression. Levels of related proteins were reduced or increased after BART22 inhibitor transfection or LY294002 treatment. GAPDH served as controls. (b) Immunohistochemistry analysis of E-cadherin, N-cadherin, Nanog and OCT4 expression. Original magnification, ×200, scale bar, 100 μm. (c) Western blotting analysis of the effect of miR-BART22 overexpression on GSK3β stability in NPC cells treated with cycloheximide and MG132 at different time points, GAPDH served as controls.

    Techniques Used: Western Blot, Over Expression, Transfection, Immunohistochemistry, Expressing

    28) Product Images from "Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer"

    Article Title: Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2016.27

    Identification of GW8510 as a potential RRM2 inhibitor. ( a ) The chemical structures of GW8510. ( b ) HCT116 cells were treated with various doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay. ( c ) HCT116 cells were treated with various doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( d ) HCT116 cells were treated with various doses of GW8510 for 24 h in the absence or presence of 5 μ M MG132. The protein expressions were analyzed by western blots. ( e ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 48 h, and then treated with indicated doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( f ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 24 h, and then treated with indicated doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay.
    Figure Legend Snippet: Identification of GW8510 as a potential RRM2 inhibitor. ( a ) The chemical structures of GW8510. ( b ) HCT116 cells were treated with various doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay. ( c ) HCT116 cells were treated with various doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( d ) HCT116 cells were treated with various doses of GW8510 for 24 h in the absence or presence of 5 μ M MG132. The protein expressions were analyzed by western blots. ( e ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 48 h, and then treated with indicated doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( f ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 24 h, and then treated with indicated doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay.

    Techniques Used: MTT Assay, Western Blot, Transfection, Plasmid Preparation

    29) Product Images from "Upregulation of Id1 by Epstein-Barr Virus-encoded LMP1 confers resistance to TGF?-mediated growth inhibition"

    Article Title: Upregulation of Id1 by Epstein-Barr Virus-encoded LMP1 confers resistance to TGF?-mediated growth inhibition

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-155

    LMP1 suppresses the expression and transcriptional activity of Foxo3a . (A) HEK-293 cells were transfected with increasing amount of the LMP1 vector (pSG5-LMP1) as indicated. Forty-eight hours post-transfection, cells were harvested for immunoblotting analysis. For the detection of Id1 and Foxo3a, cells were cultured in serum free medium for 6 hrs before harvesting. (B) Total cell lysates of NP69-pLNSX control and NP69-LMP1 cells were analyzed by immunoblotting. For detection of cytoplasmic and nuclear proteins, cells were treated with protease inhibitor, MG132 (20μM) for 4 hrs prior to harvesting. Relative protein expression was calculated using densitometry with the control set at 1. (C) HEK-293 cells were transfected with various doses of the LMP1 expression vector (pSG5-LMP1) and reporter constructs for p27 kip or Bim. (D) HEK-293 cells were transfected with various doses of GFP-Foxo3a and the p27 kip or Bim promoter reporter constructs, together with 40 ng LMP1 expression vector (pSG5-LMP1) or control empty vector (pSG5). Cells were harvested for luciferase reporter analysis 48 hrs post-transfection. Luciferase activity was normalized to β-gal activity. Data shown are the mean ± s.d. of three separate experiments. The relative luciferase unit (RLU) is plotted relative to that of the reporter alone (set at 1).
    Figure Legend Snippet: LMP1 suppresses the expression and transcriptional activity of Foxo3a . (A) HEK-293 cells were transfected with increasing amount of the LMP1 vector (pSG5-LMP1) as indicated. Forty-eight hours post-transfection, cells were harvested for immunoblotting analysis. For the detection of Id1 and Foxo3a, cells were cultured in serum free medium for 6 hrs before harvesting. (B) Total cell lysates of NP69-pLNSX control and NP69-LMP1 cells were analyzed by immunoblotting. For detection of cytoplasmic and nuclear proteins, cells were treated with protease inhibitor, MG132 (20μM) for 4 hrs prior to harvesting. Relative protein expression was calculated using densitometry with the control set at 1. (C) HEK-293 cells were transfected with various doses of the LMP1 expression vector (pSG5-LMP1) and reporter constructs for p27 kip or Bim. (D) HEK-293 cells were transfected with various doses of GFP-Foxo3a and the p27 kip or Bim promoter reporter constructs, together with 40 ng LMP1 expression vector (pSG5-LMP1) or control empty vector (pSG5). Cells were harvested for luciferase reporter analysis 48 hrs post-transfection. Luciferase activity was normalized to β-gal activity. Data shown are the mean ± s.d. of three separate experiments. The relative luciferase unit (RLU) is plotted relative to that of the reporter alone (set at 1).

    Techniques Used: Expressing, Activity Assay, Transfection, Plasmid Preparation, Cell Culture, Protease Inhibitor, Construct, Luciferase

    30) Product Images from "Synergistic stress exacerbation in hippocampal neurons: Evidence favoring the dual hit hypothesis of neurodegeneration"

    Article Title: Synergistic stress exacerbation in hippocampal neurons: Evidence favoring the dual hit hypothesis of neurodegeneration

    Journal: Hippocampus

    doi: 10.1002/hipo.22580

    N-acetyl cysteine prevents loss of MAP2 + neuronal profiles after dual MG132/MG132 hits
    Figure Legend Snippet: N-acetyl cysteine prevents loss of MAP2 + neuronal profiles after dual MG132/MG132 hits

    Techniques Used:

    Impact of dual hits of MG132 on hippocampal neuron viability
    Figure Legend Snippet: Impact of dual hits of MG132 on hippocampal neuron viability

    Techniques Used:

    31) Product Images from "Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in DrosophilaUSP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization"

    Article Title: Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in DrosophilaUSP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001239

    Smo is internalized and degraded by multi-site ubiquitination. (A) Cell extracts from S2 cells transfected with Myc-Smo, Myc-Smo K6R , Myc-Smo K7R , or Myc-Smo K13R were immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub (top) or anti-Myc antibody (bottom). (B) S2 cells were transfected with Myc-Smo or Myc-Smo K13R together with Myc-CFP (as internal control) and treated with cycloheximide (CHX) for the indicated time. Cell extracts were subjected to Western blot analysis with anti-Myc antibody. Quantification of the Western blot analysis is shown at bottom. (C) S2 cells were transfected with Myc-Smo or Myc-Smo K13R together with Myc-CFP and treated without or with MG132 and/or NH 4 Cl. Cell extracts were subjected to Western blot analysis with anti-Myc antibody. (D) S2 cells transfected with Myc-Smo or Myc-Smo K13R and treated with or without Hh-conditioned medium were immunostained with anti-SmoN antibody prior to (top panels) or after (bottom panels) membrane permeabilization. Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal.
    Figure Legend Snippet: Smo is internalized and degraded by multi-site ubiquitination. (A) Cell extracts from S2 cells transfected with Myc-Smo, Myc-Smo K6R , Myc-Smo K7R , or Myc-Smo K13R were immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub (top) or anti-Myc antibody (bottom). (B) S2 cells were transfected with Myc-Smo or Myc-Smo K13R together with Myc-CFP (as internal control) and treated with cycloheximide (CHX) for the indicated time. Cell extracts were subjected to Western blot analysis with anti-Myc antibody. Quantification of the Western blot analysis is shown at bottom. (C) S2 cells were transfected with Myc-Smo or Myc-Smo K13R together with Myc-CFP and treated without or with MG132 and/or NH 4 Cl. Cell extracts were subjected to Western blot analysis with anti-Myc antibody. (D) S2 cells transfected with Myc-Smo or Myc-Smo K13R and treated with or without Hh-conditioned medium were immunostained with anti-SmoN antibody prior to (top panels) or after (bottom panels) membrane permeabilization. Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    UBPY regulates Smo ubiquitination and cell surface expression. (A) Myc-Smo expressing cells were treated with or without Hh-conditioned medium in the presence of UBPY or Luc dsRNA. After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub or anti-Myc antibody. Of note, shorter exposure was used for Western blot analysis of samples derived from cells not treated with Hh (left). (B) S2 cells were transfected with Myc-Smo and HA-tagged Ub (HA-Ub) and with or without Flag-tagged UBPY (Fg-UBPY). After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-HA or anti-Myc antibody. (C–D) S2 cells were transfected with Fg-UBPY and Myc-tagged wild type Smo or the indicated Smo variants and treated with or without Hh-conditioned medium. Western blot analyses were carried out on cell lysates or immunoprecipitates using the indicated antibodies. Asterisks indicate monomeric forms of Myc-Smo and Myc-Smo ΔCT . (E–E”) Large magnification view of a wing disc carrying UBPY mutant clones and immunostained to show the expression of Smo (red channel) and GFP (green channel). UBPY mutant clones are marked by the lack of GFP expression. Posterior UBPY mutant clones had reduced cell surface accumulation of Smo (arrows). (F–J”) Wild type wing discs (F–F”, I–I”) or wing discs expressing UAS-UBPY alone (G–G”, J–J”) or together with UAS-Smo-RNAi (H–H”) under the control of MS1096 were immunostained to show the expression of Smo (red), Ci (green), and dpp-lacZ or ptc-lacZ (blue). (K) Confocal images of S2 cells expressing Myc-Smo (red) alone or together with Fg-UBPY (green). Top panels show cell surface staining while bottom panels show regular staining.
    Figure Legend Snippet: UBPY regulates Smo ubiquitination and cell surface expression. (A) Myc-Smo expressing cells were treated with or without Hh-conditioned medium in the presence of UBPY or Luc dsRNA. After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub or anti-Myc antibody. Of note, shorter exposure was used for Western blot analysis of samples derived from cells not treated with Hh (left). (B) S2 cells were transfected with Myc-Smo and HA-tagged Ub (HA-Ub) and with or without Flag-tagged UBPY (Fg-UBPY). After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-HA or anti-Myc antibody. (C–D) S2 cells were transfected with Fg-UBPY and Myc-tagged wild type Smo or the indicated Smo variants and treated with or without Hh-conditioned medium. Western blot analyses were carried out on cell lysates or immunoprecipitates using the indicated antibodies. Asterisks indicate monomeric forms of Myc-Smo and Myc-Smo ΔCT . (E–E”) Large magnification view of a wing disc carrying UBPY mutant clones and immunostained to show the expression of Smo (red channel) and GFP (green channel). UBPY mutant clones are marked by the lack of GFP expression. Posterior UBPY mutant clones had reduced cell surface accumulation of Smo (arrows). (F–J”) Wild type wing discs (F–F”, I–I”) or wing discs expressing UAS-UBPY alone (G–G”, J–J”) or together with UAS-Smo-RNAi (H–H”) under the control of MS1096 were immunostained to show the expression of Smo (red), Ci (green), and dpp-lacZ or ptc-lacZ (blue). (K) Confocal images of S2 cells expressing Myc-Smo (red) alone or together with Fg-UBPY (green). Top panels show cell surface staining while bottom panels show regular staining.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Derivative Assay, Transfection, Mutagenesis, Clone Assay, Staining

    Smo is regulated by both multi- and polyubiquitination. (A) S2 cells were transfected with HA-Ub K0 and Myc-Smo or indicated KR variants and treated with NH 4 Cl. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. (B) S2 cells were transfected with Myc-Smo and HA-Ub K0 or HA-Ub and treated with or without Hh-conditioned medium and/or MG132. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. The cell lysates were also immunoblotted with anti-HA antibody. (C) Myc-Smo expressing S2 cells or control cells were mock treated, or treated with either MG132 or NH 4 Cl. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc antibody or a Lys 48-linkage specific polyubiquitin antibody (K48). Of note, Loading was normalized by the amount of Myc-Smo monomer.
    Figure Legend Snippet: Smo is regulated by both multi- and polyubiquitination. (A) S2 cells were transfected with HA-Ub K0 and Myc-Smo or indicated KR variants and treated with NH 4 Cl. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. (B) S2 cells were transfected with Myc-Smo and HA-Ub K0 or HA-Ub and treated with or without Hh-conditioned medium and/or MG132. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. The cell lysates were also immunoblotted with anti-HA antibody. (C) Myc-Smo expressing S2 cells or control cells were mock treated, or treated with either MG132 or NH 4 Cl. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc antibody or a Lys 48-linkage specific polyubiquitin antibody (K48). Of note, Loading was normalized by the amount of Myc-Smo monomer.

    Techniques Used: Transfection, Immunoprecipitation, Expressing

    Smo is stabilized by both lysosome and proteasome inhibitors. (A) S2 cells stably expressing Myc-Smo were treated with MG132 and/or NH 4 Cl alone or in combination, followed by Western blot analysis with an anti-Myc antibody. (B) S2 cells stably expressing Myc-Smo treated with or without MG132 and/or Hh-conditioned medium were immunostained with anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). MG132 treatment stabilized Smo in intracellular vesicles whereas Hh treatment led to cell surface accumulation of Smo. (C) Myc-Smo expressing S2 cells were transfected with YFP tagged Rab5 or Rab7, treated with or without MG132 and immunostained to show the expression of Myc-Smo (green) and Rab5/Rab7 (red).
    Figure Legend Snippet: Smo is stabilized by both lysosome and proteasome inhibitors. (A) S2 cells stably expressing Myc-Smo were treated with MG132 and/or NH 4 Cl alone or in combination, followed by Western blot analysis with an anti-Myc antibody. (B) S2 cells stably expressing Myc-Smo treated with or without MG132 and/or Hh-conditioned medium were immunostained with anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). MG132 treatment stabilized Smo in intracellular vesicles whereas Hh treatment led to cell surface accumulation of Smo. (C) Myc-Smo expressing S2 cells were transfected with YFP tagged Rab5 or Rab7, treated with or without MG132 and immunostained to show the expression of Myc-Smo (green) and Rab5/Rab7 (red).

    Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection

    Uba1 regulates Smo ubiquitination and cell surface expression. (A–B') Low (A, B) and high (A', B') magnification view of wing imaginal discs carrying Uba1 H33 mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. Uba1 H33 mutant clones are marked by the lack of GFP staining. Arrows and arrowheads indicate anterior and posterior clones, respectively. (C) The efficiency of Uba1 RNAi was evaluated by Western blot analysis of transfected Myc-Uba1. (D) S2 cells stably expressing a Myc-tagged Smo under the control of metallothionein promoter were treated with Uba1 dsRNA or control (Luciferase) dsRNA in the absence or presence of the E1 inhibitor PYR41. After treatment with MG132, cells extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with an anti-Ub antibody to visualize ubiquitinated Smo (top) or anti-Myc antibody to visualize Myc-Smo (bottom). Loading was normalized by the amount of Myc-Smo monomer. IP, immunoprecipitation; IB, immunoblot. (E) S2 cells stably expressing Myc-Smo were treated as in (D). Cells were immunostained with anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal.
    Figure Legend Snippet: Uba1 regulates Smo ubiquitination and cell surface expression. (A–B') Low (A, B) and high (A', B') magnification view of wing imaginal discs carrying Uba1 H33 mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. Uba1 H33 mutant clones are marked by the lack of GFP staining. Arrows and arrowheads indicate anterior and posterior clones, respectively. (C) The efficiency of Uba1 RNAi was evaluated by Western blot analysis of transfected Myc-Uba1. (D) S2 cells stably expressing a Myc-tagged Smo under the control of metallothionein promoter were treated with Uba1 dsRNA or control (Luciferase) dsRNA in the absence or presence of the E1 inhibitor PYR41. After treatment with MG132, cells extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with an anti-Ub antibody to visualize ubiquitinated Smo (top) or anti-Myc antibody to visualize Myc-Smo (bottom). Loading was normalized by the amount of Myc-Smo monomer. IP, immunoprecipitation; IB, immunoblot. (E) S2 cells stably expressing Myc-Smo were treated as in (D). Cells were immunostained with anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal.

    Techniques Used: Expressing, Mutagenesis, Clone Assay, Staining, Western Blot, Transfection, Stable Transfection, Luciferase, Immunoprecipitation

    32) Product Images from "The water channel aquaporin-1 partitions into exosomes during reticulocyte maturation: implication for the regulation of cell volume"

    Article Title: The water channel aquaporin-1 partitions into exosomes during reticulocyte maturation: implication for the regulation of cell volume

    Journal: Blood

    doi: 10.1182/blood-2009-06-230086

    Inhibition of AQP-1 trafficking and sorting by MG132 . (A) Freshly obtained mouse reticulocytes (0 hour; left lane) were cultured for 24 hours with the proteasome inhibitor MG132 (20 μM; 24 hours; right lane). Freshly obtained mouse reticulocytes
    Figure Legend Snippet: Inhibition of AQP-1 trafficking and sorting by MG132 . (A) Freshly obtained mouse reticulocytes (0 hour; left lane) were cultured for 24 hours with the proteasome inhibitor MG132 (20 μM; 24 hours; right lane). Freshly obtained mouse reticulocytes

    Techniques Used: Inhibition, Cell Culture

    33) Product Images from "The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation"

    Article Title: The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2014/940186

    Effects of EGb 761 on degradation of polyQ proteins and polyQ aggregation. (a) d2GFP-HEK cells expressing htt_Q25 and (b–d) HEK293 cells expressing htt_Q103 were treated with EGb 761 or vehicle and subsequently chased with proteasome (MG132) or autophagy inhibitor (bafilomycin) to assess polyQ degradation and aggregation. (a) d2GFP-HEK cells expressing htt_Q25 were treated for 48 h with 150 μ g/mL EGb 761 or vehicle. Then, cells were incubated for 4 h with or without increasing concentrations of MG132. Protein levels of whole cell extracts were analyzed by immunoblotting to their corresponding antibodies. Protein levels of short-lived, unstable proteins (polyubiquitin, d2GFP) accumulated with proteasome inhibition while levels of long-lived polyQ proteins were not significantly altered. Values of d2GFP or polyQ protein of vehicle-treated cells without MG132 were arbitrarily set to 1; n = 4. (b-c) HEK293 cells expressing htt_Q103 were treated for 48 h with 150 μ g/mL EGb 761 or vehicle. Then, cells were subsequently incubated for 4 h with or without increasing concentrations of MG132. (b) Whole cell extracts were subjected to a filter retardation assay to assess polyQ aggregates, induced by pharmacologic proteasome inhibition. For the detection of aggregates of polyQ proteins trapped on nitrocellulose membrane an anti-eGFP antibody was used. Densitometric values of vehicle-treated cells without MG132 were arbitrarily set to 1; n = 5. (c) Protein levels of whole cell extracts (samples from b) were analyzed by immunoblotting to their corresponding antibodies. Protein levels of unstable, misfolded polyQ proteins accumulated with proteasome inhibition while levels of stable, soluble polyQ proteins were not altered. Values of soluble or insoluble polyQ proteins of vehicle-treated cells without MG132 were arbitrarily set to 1; n = 3. (d) HEK293 cells expressing htt_Q103 were treated for 48 h with 150 μ g/mL EGb 761 or vehicle. Then, cells were subsequently incubated for 3 h with or without 1 μ M of the lysosomal inhibitor bafilomycin A1 (denoted bafi.) to investigate the autophagic flux. Whole cell extracts were analyzed by immunoblotting or filter retardation assay. Cells treated with EGb 761 showed no significant alteration with bafilomycin treatment in protein levels of LC3-I to LC3-II, soluble and insoluble polyQ, indicating no direct effect on autophagy by EGb 761. Immunoblotting and filter retardation assay confirmed significant changes in aggregated polyQ proteins by EGb 761 from previous experiments (Figures 3(b) – 3(d) and Figures 4(b) - 4(c) ). Densitometric values of vehicle-treated cells without bafilomycin were arbitrarily set to 1; n = 3. (a–d) All values are reported as mean ± S.D. * P
    Figure Legend Snippet: Effects of EGb 761 on degradation of polyQ proteins and polyQ aggregation. (a) d2GFP-HEK cells expressing htt_Q25 and (b–d) HEK293 cells expressing htt_Q103 were treated with EGb 761 or vehicle and subsequently chased with proteasome (MG132) or autophagy inhibitor (bafilomycin) to assess polyQ degradation and aggregation. (a) d2GFP-HEK cells expressing htt_Q25 were treated for 48 h with 150 μ g/mL EGb 761 or vehicle. Then, cells were incubated for 4 h with or without increasing concentrations of MG132. Protein levels of whole cell extracts were analyzed by immunoblotting to their corresponding antibodies. Protein levels of short-lived, unstable proteins (polyubiquitin, d2GFP) accumulated with proteasome inhibition while levels of long-lived polyQ proteins were not significantly altered. Values of d2GFP or polyQ protein of vehicle-treated cells without MG132 were arbitrarily set to 1; n = 4. (b-c) HEK293 cells expressing htt_Q103 were treated for 48 h with 150 μ g/mL EGb 761 or vehicle. Then, cells were subsequently incubated for 4 h with or without increasing concentrations of MG132. (b) Whole cell extracts were subjected to a filter retardation assay to assess polyQ aggregates, induced by pharmacologic proteasome inhibition. For the detection of aggregates of polyQ proteins trapped on nitrocellulose membrane an anti-eGFP antibody was used. Densitometric values of vehicle-treated cells without MG132 were arbitrarily set to 1; n = 5. (c) Protein levels of whole cell extracts (samples from b) were analyzed by immunoblotting to their corresponding antibodies. Protein levels of unstable, misfolded polyQ proteins accumulated with proteasome inhibition while levels of stable, soluble polyQ proteins were not altered. Values of soluble or insoluble polyQ proteins of vehicle-treated cells without MG132 were arbitrarily set to 1; n = 3. (d) HEK293 cells expressing htt_Q103 were treated for 48 h with 150 μ g/mL EGb 761 or vehicle. Then, cells were subsequently incubated for 3 h with or without 1 μ M of the lysosomal inhibitor bafilomycin A1 (denoted bafi.) to investigate the autophagic flux. Whole cell extracts were analyzed by immunoblotting or filter retardation assay. Cells treated with EGb 761 showed no significant alteration with bafilomycin treatment in protein levels of LC3-I to LC3-II, soluble and insoluble polyQ, indicating no direct effect on autophagy by EGb 761. Immunoblotting and filter retardation assay confirmed significant changes in aggregated polyQ proteins by EGb 761 from previous experiments (Figures 3(b) – 3(d) and Figures 4(b) - 4(c) ). Densitometric values of vehicle-treated cells without bafilomycin were arbitrarily set to 1; n = 3. (a–d) All values are reported as mean ± S.D. * P

    Techniques Used: Expressing, Incubation, Inhibition

    Effects of EGb 761 on basal proteasome activity. (a) HEK293 cells were treated for 24 h with indicated concentrations of EGb 761. Analysis of proteasomal peptidase (chymotrypsin-like) activity was assessed by the hydrolysis of SUC-LLVY-AMC in total cell lysates. Fluorescence of the cleaved AMC moiety was measured in the presence or absence of MG132 to achieve peptidase specificity. Values were adjusted to total protein content. Activity of vehicle-treated cells was arbitrarily set to 1; n = 4. (b–d) HEK293 cells with stable expressions of the proteasome reporter protein d2GFP (d2GFP-HEK) were treated for 24 h with indicated concentrations of EGb 761. Measurement of GFP fluorescence was used to investigate proteasomal degradation of d2GFP proteins. All achieved fluorescence intensities were finally adjusted to total protein content. (b) Cells were incubated with increasing concentrations of EGb 761 to investigate the specific, dose-depending effect on proteasome activity (a). Measurement of GFP fluorescence was used to assess the remaining d2GFP protein content as an indicator for an enhanced protein degradation by the proteasome. Values of vehicle-treated cells were arbitrarily set to 1. n = 5. (c) Previous assayed cells ((b); vehicle and 150 μ g/mL EGb 761 treatments) were additionally incubated for 2 h with the proteasome inhibitor MG132 or DMSO as control. The addition of MG132 led to an increase of fluorescence intensities in control and EGb 761-treated cells. Inhibition of proteasome activity showed the specific modulation of GFP fluorescence through proteasomal d2GFP degradation. Values of vehicle-treated cells without MG132 were arbitrarily set to 1. n = 4. (d) Cells were treated for 24 h with 150 μ g/mL EGb 761 or vehicle, followed by a chase with cycloheximide (CHX) to block synthesis of new d2GFP. Degradation kinetics of d2GFP was analyzed by measuring GFP fluorescence every 30 min. Fluorescence decay induced by CHX indicated the specificity of proteasomal d2GFP degradation. Values of each treatment at zero minutes were arbitrarily set to 1; n = 3. (e) HEK293 cells were treated for 2 h with 150 μ g/mL EGb 761 or vehicle and RNA was extracted for qRT-PCR analysis. Relative expression ratio of proteasome genes PSMB5, PSMB6, and PSMB7 in EGb 761-treated cells to vehicle-treated cells is shown; n = 3. (a–e) All values are reported as mean ± S.D. * P
    Figure Legend Snippet: Effects of EGb 761 on basal proteasome activity. (a) HEK293 cells were treated for 24 h with indicated concentrations of EGb 761. Analysis of proteasomal peptidase (chymotrypsin-like) activity was assessed by the hydrolysis of SUC-LLVY-AMC in total cell lysates. Fluorescence of the cleaved AMC moiety was measured in the presence or absence of MG132 to achieve peptidase specificity. Values were adjusted to total protein content. Activity of vehicle-treated cells was arbitrarily set to 1; n = 4. (b–d) HEK293 cells with stable expressions of the proteasome reporter protein d2GFP (d2GFP-HEK) were treated for 24 h with indicated concentrations of EGb 761. Measurement of GFP fluorescence was used to investigate proteasomal degradation of d2GFP proteins. All achieved fluorescence intensities were finally adjusted to total protein content. (b) Cells were incubated with increasing concentrations of EGb 761 to investigate the specific, dose-depending effect on proteasome activity (a). Measurement of GFP fluorescence was used to assess the remaining d2GFP protein content as an indicator for an enhanced protein degradation by the proteasome. Values of vehicle-treated cells were arbitrarily set to 1. n = 5. (c) Previous assayed cells ((b); vehicle and 150 μ g/mL EGb 761 treatments) were additionally incubated for 2 h with the proteasome inhibitor MG132 or DMSO as control. The addition of MG132 led to an increase of fluorescence intensities in control and EGb 761-treated cells. Inhibition of proteasome activity showed the specific modulation of GFP fluorescence through proteasomal d2GFP degradation. Values of vehicle-treated cells without MG132 were arbitrarily set to 1. n = 4. (d) Cells were treated for 24 h with 150 μ g/mL EGb 761 or vehicle, followed by a chase with cycloheximide (CHX) to block synthesis of new d2GFP. Degradation kinetics of d2GFP was analyzed by measuring GFP fluorescence every 30 min. Fluorescence decay induced by CHX indicated the specificity of proteasomal d2GFP degradation. Values of each treatment at zero minutes were arbitrarily set to 1; n = 3. (e) HEK293 cells were treated for 2 h with 150 μ g/mL EGb 761 or vehicle and RNA was extracted for qRT-PCR analysis. Relative expression ratio of proteasome genes PSMB5, PSMB6, and PSMB7 in EGb 761-treated cells to vehicle-treated cells is shown; n = 3. (a–e) All values are reported as mean ± S.D. * P

    Techniques Used: Activity Assay, Fluorescence, Incubation, Inhibition, Blocking Assay, Quantitative RT-PCR, Expressing

    Modulation of proteasome activity by EGb 761 in cells expressing polyQ proteins. (a, c-d) HEK293 or (b) d2GFP-HEK cells were transiently transfected to express the polyQ fusion proteins with glutamine expansions of 25 (htt_Q25), 46 (htt_Q46), and 103 repeats (htt_Q103). Subsequent to settlement for 24 h cells were treated with 150 μ g/mL EGb 761 or vehicle for different time periods and further investigated. (a) HEK293 cells expressing eGFP and polyQ proteins (htt_Q25, Q46, and Q103) were treated for 24 h with EGb 761 or vehicle, followed by analysis of proteasomal peptidase activity in total cell lysates. Fluorescence of AMC moiety resulting from peptidase hydrolysis of SUC-LLVY-AMC was measured in the presence or absence of MG132 to achieve peptidase specificity. Values were adjusted to total protein content. Activity of vehicle-treated eGFP-expressing cells was arbitrarily set to 1; n = 3. (b) d2GFP-HEK cells expressing polyQ proteins (htt_Q25, Q46, and Q103) were treated for 24 h with EGb 761 or vehicle. Cell lysates of each sample were collected and analyzed by immunoblotting. Protein levels of d2GFP and proteasome subunit α 1 were analyzed with their corresponding antibody to assess proteasome activity and status. Densitometric values of vehicle-treated and htt_Q25-expressing cells were arbitrarily set to 1; n = 4. (c-d) HEK293 cells expressing htt_Q25 and htt_Q103 for 24 h were analyzed by qRT-PCR for transcript levels of PSMB5, PSMB6, and PSMB7. (c) Quantification of basal expression ratio in cells expressing htt_Q103 to htt_Q25 (without EGb 761 treatment). (d) Analysis of cells additionally treated with EGb 761 or vehicle for 2 h. Expression ratio of proteasome genes in cells treated with EGb 761 compared to vehicle; n = 3. (a–d) All values are reported as mean ± S.D. * P
    Figure Legend Snippet: Modulation of proteasome activity by EGb 761 in cells expressing polyQ proteins. (a, c-d) HEK293 or (b) d2GFP-HEK cells were transiently transfected to express the polyQ fusion proteins with glutamine expansions of 25 (htt_Q25), 46 (htt_Q46), and 103 repeats (htt_Q103). Subsequent to settlement for 24 h cells were treated with 150 μ g/mL EGb 761 or vehicle for different time periods and further investigated. (a) HEK293 cells expressing eGFP and polyQ proteins (htt_Q25, Q46, and Q103) were treated for 24 h with EGb 761 or vehicle, followed by analysis of proteasomal peptidase activity in total cell lysates. Fluorescence of AMC moiety resulting from peptidase hydrolysis of SUC-LLVY-AMC was measured in the presence or absence of MG132 to achieve peptidase specificity. Values were adjusted to total protein content. Activity of vehicle-treated eGFP-expressing cells was arbitrarily set to 1; n = 3. (b) d2GFP-HEK cells expressing polyQ proteins (htt_Q25, Q46, and Q103) were treated for 24 h with EGb 761 or vehicle. Cell lysates of each sample were collected and analyzed by immunoblotting. Protein levels of d2GFP and proteasome subunit α 1 were analyzed with their corresponding antibody to assess proteasome activity and status. Densitometric values of vehicle-treated and htt_Q25-expressing cells were arbitrarily set to 1; n = 4. (c-d) HEK293 cells expressing htt_Q25 and htt_Q103 for 24 h were analyzed by qRT-PCR for transcript levels of PSMB5, PSMB6, and PSMB7. (c) Quantification of basal expression ratio in cells expressing htt_Q103 to htt_Q25 (without EGb 761 treatment). (d) Analysis of cells additionally treated with EGb 761 or vehicle for 2 h. Expression ratio of proteasome genes in cells treated with EGb 761 compared to vehicle; n = 3. (a–d) All values are reported as mean ± S.D. * P

    Techniques Used: Activity Assay, Expressing, Transfection, Fluorescence, Quantitative RT-PCR

    34) Product Images from "Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling"

    Article Title: Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling

    Journal: eLife

    doi: 10.7554/eLife.23968

    iRhom2 interaction with TACE, and further characterisation of the deletion mutants of iRhom2. ( a ) HA immunoprecipitates (IP) and lysates (lys) from DKO MEFs transduced with GFP or HA-tagged iRhom2 WT , immunoblotted for HA, TACE and beta-actin. Empty beads (without conjugated HA antibody) are used to show the specificity of the co-immunoprecipitation. ( b ) Lysates (lys) and neutravidin-enriched preparations (biotin PD) from MEFs transduced with GFP, or HA-tagged iRhom2 WT , iRhom2 ∆NT and iRhom2 ∆IRHD labelled with biotin and probed with HA and transferrin receptor antibodies. ( c ) Lysates from DKO MEFs expressing HA-tagged iRhom2 WT and iRhom2 ∆NT enriched for glycoproteins with ConA were immunoblotted for TACE, HA, and transferrin receptor (TfR) as a labelling control. Where indicated, cells had been treated with the lysosomal degradation inhibitor bafilomycin A1 (Baf A, 100 nM) or the proteosomal degradation inhibitor MG132 (10 µM) for 4 hr. Lysates were probed for beta-actin, and immunoblotted for ubiquitin to demonstrate the efficacy of MG132 treatment. DOI: http://dx.doi.org/10.7554/eLife.23968.010
    Figure Legend Snippet: iRhom2 interaction with TACE, and further characterisation of the deletion mutants of iRhom2. ( a ) HA immunoprecipitates (IP) and lysates (lys) from DKO MEFs transduced with GFP or HA-tagged iRhom2 WT , immunoblotted for HA, TACE and beta-actin. Empty beads (without conjugated HA antibody) are used to show the specificity of the co-immunoprecipitation. ( b ) Lysates (lys) and neutravidin-enriched preparations (biotin PD) from MEFs transduced with GFP, or HA-tagged iRhom2 WT , iRhom2 ∆NT and iRhom2 ∆IRHD labelled with biotin and probed with HA and transferrin receptor antibodies. ( c ) Lysates from DKO MEFs expressing HA-tagged iRhom2 WT and iRhom2 ∆NT enriched for glycoproteins with ConA were immunoblotted for TACE, HA, and transferrin receptor (TfR) as a labelling control. Where indicated, cells had been treated with the lysosomal degradation inhibitor bafilomycin A1 (Baf A, 100 nM) or the proteosomal degradation inhibitor MG132 (10 µM) for 4 hr. Lysates were probed for beta-actin, and immunoblotted for ubiquitin to demonstrate the efficacy of MG132 treatment. DOI: http://dx.doi.org/10.7554/eLife.23968.010

    Techniques Used: Transduction, Immunoprecipitation, Expressing

    35) Product Images from "A highly conserved redox-active Mx(2)CWx(6)R motif regulates Zap70 stability and activity"

    Article Title: A highly conserved redox-active Mx(2)CWx(6)R motif regulates Zap70 stability and activity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16486

    Analysis of protein stability of Zap70C575A A . Representative histograms of the expression of Zap70 wt and C575A. P116 cells were transfected with YFP-tagged Zap70wt or C575A. Transfection efficiency was determined by gating on YFP + cells. B . Zap70wt or C575A expressing P116 cells were lysed and subjected to immunoblot analysis with an antibody against human Zap70. C . P116 cells reconstituted with either Zap70wt-YFP or C575A-YFP were either left untreated or treated with 20μg/mL cycloheximide for different time points up to 8h. Zap70 protein expression was measured by flow cytometry. The values of wt and mutant Zap70-YFP MFI were normalized to the respective untreated control samples for each time point followed by normalization to the 0h time point which was set to 1 for both wt and mutant Zap70 ( n = 3). D . HEK293T cells were transfected with Zap70-Myc-DDK or C575A-Myc-DDK. After starvation of cysteine and methionine, cells were pulsed with [S35]methionine/cysteine for 15min, followed by chase up to 6h. Subsequently, cells were lysed and Zap70 was immunoprecipitated using anti-FLAG antibody. Samples were separated using SDS-PAGE and signals were detected by autoradiography and quantified by densitometry ( n = 2). E . Zap70wt-YFP of C575A-YFP expressing P116 cells were either treated with 20mM MG132 (+MG132) or DMSO (-MG132) for 4h. Subsequently, protein expression was measured by flow cytometry. YFP MFI values of wt and mutant Zap70 were normalized to the respective untreated control samples which were set to 1 ( n = 4).
    Figure Legend Snippet: Analysis of protein stability of Zap70C575A A . Representative histograms of the expression of Zap70 wt and C575A. P116 cells were transfected with YFP-tagged Zap70wt or C575A. Transfection efficiency was determined by gating on YFP + cells. B . Zap70wt or C575A expressing P116 cells were lysed and subjected to immunoblot analysis with an antibody against human Zap70. C . P116 cells reconstituted with either Zap70wt-YFP or C575A-YFP were either left untreated or treated with 20μg/mL cycloheximide for different time points up to 8h. Zap70 protein expression was measured by flow cytometry. The values of wt and mutant Zap70-YFP MFI were normalized to the respective untreated control samples for each time point followed by normalization to the 0h time point which was set to 1 for both wt and mutant Zap70 ( n = 3). D . HEK293T cells were transfected with Zap70-Myc-DDK or C575A-Myc-DDK. After starvation of cysteine and methionine, cells were pulsed with [S35]methionine/cysteine for 15min, followed by chase up to 6h. Subsequently, cells were lysed and Zap70 was immunoprecipitated using anti-FLAG antibody. Samples were separated using SDS-PAGE and signals were detected by autoradiography and quantified by densitometry ( n = 2). E . Zap70wt-YFP of C575A-YFP expressing P116 cells were either treated with 20mM MG132 (+MG132) or DMSO (-MG132) for 4h. Subsequently, protein expression was measured by flow cytometry. YFP MFI values of wt and mutant Zap70 were normalized to the respective untreated control samples which were set to 1 ( n = 4).

    Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry, Mutagenesis, Immunoprecipitation, SDS Page, Autoradiography

    36) Product Images from "LncRNA HOTAIR regulates lipopolysaccharide-induced cytokine expression and inflammatory response in macrophages"

    Article Title: LncRNA HOTAIR regulates lipopolysaccharide-induced cytokine expression and inflammatory response in macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33722-2

    HOTAIR promotes IκBα degradation and nuclear translocation of NF-κB. HOTAIR was silenced in RAW 264.7 cells by using HOTAIR antisense and scramble (control) for 48 h. Additionally, cells were also treated with proteasomal inhibitor MG132 (2 h) alone or in combination with HOTAIR-knockdown and then treated with LPS (1 h). The cells were then fixed with paraformaldehyde and immunostained with antibody against IκBα and phospho NF-κB (P-p65), and counterstained with DAPI. Images were taken by fluorescence microscope (Nikon ECLIPSE TE2000-U) ( A ) and fluorescence intensity showing the expressions of IκBα and phospho-p65 was quantified and plotted by ImageJ software ( B ). Data represent mean ± SD; *p
    Figure Legend Snippet: HOTAIR promotes IκBα degradation and nuclear translocation of NF-κB. HOTAIR was silenced in RAW 264.7 cells by using HOTAIR antisense and scramble (control) for 48 h. Additionally, cells were also treated with proteasomal inhibitor MG132 (2 h) alone or in combination with HOTAIR-knockdown and then treated with LPS (1 h). The cells were then fixed with paraformaldehyde and immunostained with antibody against IκBα and phospho NF-κB (P-p65), and counterstained with DAPI. Images were taken by fluorescence microscope (Nikon ECLIPSE TE2000-U) ( A ) and fluorescence intensity showing the expressions of IκBα and phospho-p65 was quantified and plotted by ImageJ software ( B ). Data represent mean ± SD; *p

    Techniques Used: Translocation Assay, Fluorescence, Microscopy, Software

    Related Articles

    Incubation:

    Article Title: Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity
    Article Snippet: For longer term incubation, the oocytes were cultured in M16 in a 5% CO2 humidified incubator at 37°C. .. Treatments For treatments, oocytes were incubated in culture media (M2 or M16) containing the following drugs: 50 µM roscovitine (EMD Millipore), 50 µM UO126 (Promega), 50 µM MG132 (EMD Millipore), 1 µM okadaic acid (EMD Millipore), or 2 µM AZ3146 (Santa Cruz Biotechnology, Inc.). .. Because roscovitine is oil soluble, no mineral oil was added to the media, and the culture dish was covered with a lid to prevent evaporation.

    Article Title: RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells ▿RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells ▿ †
    Article Snippet: Ninety-six-well plates were seeded with Vero E6 cells (104 ), and the cells were transduced with vAcL and either wt or vAcIE2 baculovirus at an MOI of 50. .. After overnight incubation, fresh medium containing 5 μm of MG132 (Sigma) was used to replace the virus-containing medium. .. The cells were incubated for a further 48 h before the luciferase assay.

    Article Title: Inhibition of Proteasomal Activity Causes Inclusion Formation in Neuronal and Non-Neuronal Cells Overexpressing Parkin
    Article Snippet: Therefore, we studied the localization of FLAG-HHARI in cells grown in the presence of MG132 to see whether inclusion formation was a general phenomenon of RIR domain proteins. .. For these experiments, cells were incubated with MG132 for either 4 or 16 h to assess the rate of inclusion formation. ..

    Concentration Assay:

    Article Title: Porcine Epidemic Diarrhea Virus nsp15 Antagonizes Interferon Signaling by RNA Degradation of TBK1 and IRF3
    Article Snippet: .. Some cell samples were treated with proteasome inhibitor MG132 (Sigma) at the concentration of 2 µM, autophagy inhibitor 3-Methyladenine (3-MA, Sigma) at 5 mM, or carrier control DMSO during some transfection assays as previously described [ ]. .. TransfectionHEK293 cells were transfected with indicated plasmids using X-tremeGENE transfection reagent according to manufacturer’s instruction (Roche, USA).

    Transfection:

    Article Title: Porcine Epidemic Diarrhea Virus nsp15 Antagonizes Interferon Signaling by RNA Degradation of TBK1 and IRF3
    Article Snippet: .. Some cell samples were treated with proteasome inhibitor MG132 (Sigma) at the concentration of 2 µM, autophagy inhibitor 3-Methyladenine (3-MA, Sigma) at 5 mM, or carrier control DMSO during some transfection assays as previously described [ ]. .. TransfectionHEK293 cells were transfected with indicated plasmids using X-tremeGENE transfection reagent according to manufacturer’s instruction (Roche, USA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Millipore mg 132
    <t>MG-132</t> induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Mg 132, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mg 132/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mg 132 - by Bioz Stars, 2021-07
    97/100 stars
      Buy from Supplier

    99
    Millipore bay11 7082
    MPs released by LPS-treated THP-1 cells induce the expression of cell adhesion molecules by HUVECs. (A) HUVECs were incubated for 1 or 2 hours in serum-free conditions with MPs from untreated or LPS-treated THP-1 cells or 10 ng/mL TNF-α. ICAM-1 , VCAM-1 , and E-selectin mRNA expression was measured by real-time polymerase chain reaction. (B) HUVECs were incubated for 4 hours in serum-free conditions with MPs derived from LPS-treated or untreated THP-1 cells or 10 ng/mL TNF-α. Expression of VCAM-1, E-selectin, and ICAM-1 by HUVECs was detected by Western blot. (C) HUVECs were incubated for 4 hours in serum-free conditions with MPs from LPS-treated PBMCs or 10 ng/mL TNF-α. VCAM-1 was detected by Western blot. (D) HUVECs were treated with 5 or 10μM of the NF-κB pathway inhibitors MG-132, <t>Bay11–7082,</t> or vehicle control (dimethyl sulfoxide [DMSO]) and then stimulated with MPs from LPS-treated THP-1 cells. VCAM-1 was detected by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments.
    Bay11 7082, 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/bay11 7082/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bay11 7082 - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    99
    Millipore mg132
    Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM <t>MG132</t> (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p
    Mg132, 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/mg132/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mg132 - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Western Blot, Software, Transfection

    MPs released by LPS-treated THP-1 cells induce the expression of cell adhesion molecules by HUVECs. (A) HUVECs were incubated for 1 or 2 hours in serum-free conditions with MPs from untreated or LPS-treated THP-1 cells or 10 ng/mL TNF-α. ICAM-1 , VCAM-1 , and E-selectin mRNA expression was measured by real-time polymerase chain reaction. (B) HUVECs were incubated for 4 hours in serum-free conditions with MPs derived from LPS-treated or untreated THP-1 cells or 10 ng/mL TNF-α. Expression of VCAM-1, E-selectin, and ICAM-1 by HUVECs was detected by Western blot. (C) HUVECs were incubated for 4 hours in serum-free conditions with MPs from LPS-treated PBMCs or 10 ng/mL TNF-α. VCAM-1 was detected by Western blot. (D) HUVECs were treated with 5 or 10μM of the NF-κB pathway inhibitors MG-132, Bay11–7082, or vehicle control (dimethyl sulfoxide [DMSO]) and then stimulated with MPs from LPS-treated THP-1 cells. VCAM-1 was detected by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments.

    Journal: Blood

    Article Title: Monocytic microparticles activate endothelial cells in an IL-1?-dependent manner

    doi: 10.1182/blood-2011-01-330878

    Figure Lengend Snippet: MPs released by LPS-treated THP-1 cells induce the expression of cell adhesion molecules by HUVECs. (A) HUVECs were incubated for 1 or 2 hours in serum-free conditions with MPs from untreated or LPS-treated THP-1 cells or 10 ng/mL TNF-α. ICAM-1 , VCAM-1 , and E-selectin mRNA expression was measured by real-time polymerase chain reaction. (B) HUVECs were incubated for 4 hours in serum-free conditions with MPs derived from LPS-treated or untreated THP-1 cells or 10 ng/mL TNF-α. Expression of VCAM-1, E-selectin, and ICAM-1 by HUVECs was detected by Western blot. (C) HUVECs were incubated for 4 hours in serum-free conditions with MPs from LPS-treated PBMCs or 10 ng/mL TNF-α. VCAM-1 was detected by Western blot. (D) HUVECs were treated with 5 or 10μM of the NF-κB pathway inhibitors MG-132, Bay11–7082, or vehicle control (dimethyl sulfoxide [DMSO]) and then stimulated with MPs from LPS-treated THP-1 cells. VCAM-1 was detected by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments.

    Article Snippet: LPS ( Escherichia coli serotype O111:B4), MG-132, and Bay11–7082 were obtained from Sigma-Aldrich.

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot

    Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM MG132 (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p

    Journal: Scientific Reports

    Article Title: Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    doi: 10.1038/srep40825

    Figure Lengend Snippet: Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM MG132 (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p

    Article Snippet: Where indicated, cells were treated with ALLN (Sigma), brefeldin A (Sigma), chloroquine (Sigma), cycloheximide (Sigma), MG132 (Sigma), MLN4924 (Dr. Kuo-How Huang, National Taiwan University Hospital), or tunicamycin (Sigma).

    Techniques: Inhibition, Expressing, Molecular Weight, Modification, Western Blot

    Pharmacological suppression of proteasomal and lysosomal degradations alters subcellular localization of rEag1 in HEK293T cells. Representative confocal micrographs showing rEag1 immunofluorescence signals (green) in response to Cul7 co-expression ( A , B , C ), or 6-hour MG132 ( D , E , F ) or chloroquine ( G , H , I ) treatment. rEag1 and Myc-Cul7 were detected with anti-rEag1 and anti-Myc antibodies, respectively. Nuclei were counterstained with DAPI (blue). To verify plasma membrane localization, some cells were co-transfected with the DsRed-membrane expression vector (DsRed-Mem). ER and lysosomal localizations were detected by specific antibodies for the ER marker calnexin and the lysosome marker lamp1, respectively. Merge images are shown in the third column of each panel. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. See Supplementary Methods and Supplementary Figure S2B for further quantitative analyses. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    doi: 10.1038/srep40825

    Figure Lengend Snippet: Pharmacological suppression of proteasomal and lysosomal degradations alters subcellular localization of rEag1 in HEK293T cells. Representative confocal micrographs showing rEag1 immunofluorescence signals (green) in response to Cul7 co-expression ( A , B , C ), or 6-hour MG132 ( D , E , F ) or chloroquine ( G , H , I ) treatment. rEag1 and Myc-Cul7 were detected with anti-rEag1 and anti-Myc antibodies, respectively. Nuclei were counterstained with DAPI (blue). To verify plasma membrane localization, some cells were co-transfected with the DsRed-membrane expression vector (DsRed-Mem). ER and lysosomal localizations were detected by specific antibodies for the ER marker calnexin and the lysosome marker lamp1, respectively. Merge images are shown in the third column of each panel. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. See Supplementary Methods and Supplementary Figure S2B for further quantitative analyses. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least 3 independent experiments.

    Article Snippet: Where indicated, cells were treated with ALLN (Sigma), brefeldin A (Sigma), chloroquine (Sigma), cycloheximide (Sigma), MG132 (Sigma), MLN4924 (Dr. Kuo-How Huang, National Taiwan University Hospital), or tunicamycin (Sigma).

    Techniques: Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Marker, Staining

    Ataxin-3 targets mutant SOD1 to aggresomes to with autophagic activity. A , 293 cells co-expressing RFP-tagged SOD1 G85R , EGFP-tagged ataxin-3 and Flag-tagged p62 were incubated in 10 μ m MG132 for 12 h. The cells were then fixed and stained with

    Journal: The Journal of Biological Chemistry

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    doi: 10.1074/jbc.M111.299990

    Figure Lengend Snippet: Ataxin-3 targets mutant SOD1 to aggresomes to with autophagic activity. A , 293 cells co-expressing RFP-tagged SOD1 G85R , EGFP-tagged ataxin-3 and Flag-tagged p62 were incubated in 10 μ m MG132 for 12 h. The cells were then fixed and stained with

    Article Snippet: MG132 was purchased from Calbiochem.

    Techniques: Mutagenesis, Activity Assay, Expressing, Incubation, Staining

    Colocalization of mutant SOD1 and ataxin-3 in aggresomes. A , 293 cells were transfected with EGFP or SOD1 G85R -EGFP and treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were immunoprecipitated with rabbit polyclonal antibodies

    Journal: The Journal of Biological Chemistry

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    doi: 10.1074/jbc.M111.299990

    Figure Lengend Snippet: Colocalization of mutant SOD1 and ataxin-3 in aggresomes. A , 293 cells were transfected with EGFP or SOD1 G85R -EGFP and treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were immunoprecipitated with rabbit polyclonal antibodies

    Article Snippet: MG132 was purchased from Calbiochem.

    Techniques: Mutagenesis, Transfection, Immunoprecipitation

    Ataxin-3-mediated aggresome formation by mutant SOD1 depends on ataxin-3 DUB activity. A , 293 cells were co-transfected with SOD1 G85R -EGFP and HA or HA-ataxin-3, then treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were

    Journal: The Journal of Biological Chemistry

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    doi: 10.1074/jbc.M111.299990

    Figure Lengend Snippet: Ataxin-3-mediated aggresome formation by mutant SOD1 depends on ataxin-3 DUB activity. A , 293 cells were co-transfected with SOD1 G85R -EGFP and HA or HA-ataxin-3, then treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were

    Article Snippet: MG132 was purchased from Calbiochem.

    Techniques: Mutagenesis, Activity Assay, Transfection