Structured Review

Amresco leupeptin
Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or <t>leupeptin</t> (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p
Leupeptin, supplied by Amresco, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/leupeptin/product/Amresco
Average 93 stars, based on 21 article reviews
Price from $9.99 to $1999.99
leupeptin - by Bioz Stars, 2020-07
93/100 stars

Images

1) Product Images from "Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence"

Article Title: Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Journal: Autophagy

doi: 10.1080/15548627.2016.1247143

Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or leupeptin (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p
Figure Legend Snippet: Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or leupeptin (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p

Techniques Used: Fluorescence, Staining, Expressing, Quantitative RT-PCR, Transfection, shRNA, Western Blot, Cell Culture, Incubation

2) Product Images from "c-MYC responds to glucose deprivation in a cell-type-dependent manner"

Article Title: c-MYC responds to glucose deprivation in a cell-type-dependent manner

Journal: Cell Death Discovery

doi: 10.1038/cddiscovery.2015.57

Glucose deprivation differentially affects c-MYC protein stability in HeLa and MDA-MB-231 cells. ( a ) Western blot detection of c-MYC in HeLa and MDA-MB-231 cells treated with CHX (0.1 mM) and MG-132 (10 μ M) in the presence or absence of 25 mM glucose for 12 h. ( b ) HEK293T cells cotransfected with c-MYC-Flag and HA-Ub were treated with MG-132 in the presence or absence of glucose for 12 h. Ubiquitination of c-MYC was determined. ( c ) Western blot detection of c-MYC in HeLa cells treated with Bafilomycin A1 (Baf) (500 nM), Leupeptin (10 μ g/ml) or 3-MA (1 mM) in the presence or absence of glucose for 12 h. ( d ) HeLa cells were treated with CHX for the indicated time in the presence or absence of glucose. Representative immunoblots of c-MYC in three independent experiments were shown. Bottom, quantification of the c-MYC levels was shown. ( e and f ) HeLa ( e ) and MDA-MB-231 ( f ) stable cells ectopically expressing c-MYC-Flag were treated with MG-132 in the presence or absence of glucose. Exogenous c-MYC stability was examined. ( g ) Western blot detection of c-MYC in HeLa stable cells expressing WT HA-SKP2 or HA-tagged dominant negative mutant HA-SKP2(ΔF-box) under GD condition for 12 h. ( h ) HEK293T cells were transfected with WT-c-MYC-Flag or c-MYC(T58A)-Flag. Cell lysates were subjected to immunoprecipitation and phosphorylation of c-MYC was examined. ( i ) Western blot detection of c-MYC in HeLa stable cells expressing FBXW7 treated as in g .
Figure Legend Snippet: Glucose deprivation differentially affects c-MYC protein stability in HeLa and MDA-MB-231 cells. ( a ) Western blot detection of c-MYC in HeLa and MDA-MB-231 cells treated with CHX (0.1 mM) and MG-132 (10 μ M) in the presence or absence of 25 mM glucose for 12 h. ( b ) HEK293T cells cotransfected with c-MYC-Flag and HA-Ub were treated with MG-132 in the presence or absence of glucose for 12 h. Ubiquitination of c-MYC was determined. ( c ) Western blot detection of c-MYC in HeLa cells treated with Bafilomycin A1 (Baf) (500 nM), Leupeptin (10 μ g/ml) or 3-MA (1 mM) in the presence or absence of glucose for 12 h. ( d ) HeLa cells were treated with CHX for the indicated time in the presence or absence of glucose. Representative immunoblots of c-MYC in three independent experiments were shown. Bottom, quantification of the c-MYC levels was shown. ( e and f ) HeLa ( e ) and MDA-MB-231 ( f ) stable cells ectopically expressing c-MYC-Flag were treated with MG-132 in the presence or absence of glucose. Exogenous c-MYC stability was examined. ( g ) Western blot detection of c-MYC in HeLa stable cells expressing WT HA-SKP2 or HA-tagged dominant negative mutant HA-SKP2(ΔF-box) under GD condition for 12 h. ( h ) HEK293T cells were transfected with WT-c-MYC-Flag or c-MYC(T58A)-Flag. Cell lysates were subjected to immunoprecipitation and phosphorylation of c-MYC was examined. ( i ) Western blot detection of c-MYC in HeLa stable cells expressing FBXW7 treated as in g .

Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, Dominant Negative Mutation, Transfection, Immunoprecipitation

3) Product Images from "JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells"

Article Title: JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.12374

JWA is required for degradation of the HER2 protein ( A ) The SGC-7901 and NCI-N87 cells were transfected with Flag-JWA or JWA siRNA for 48 h. Western blot showed the expressions of HER2 and JWA. ( B and C ) SGC-7901 cells were transfected with JWA siRNA for 48 h, followed by treatment with 178 μM of CHX at indicated times. The protein stability of HER2 was assessed by western blotting (B). Quantification of HER2 protein levels (C). ( D ) The SGC-7901 cells were transfected with JWA siRNA, His-Ub and HER2-WT for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. The NCI-N87 cells were transfected with Flag-JWA and His-Ub for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. Western blotting was performed to confirm the levels of HER2 and JWA. Immunoprecipitation was used to show the ubiquitination of HER2. ( E and F ) SGC-7901 cells transfected with JWA siRNA or NCI-N87 cells transfected with Flag-JWA plasmid for 24 h, then incubated with or without leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of HER2 and JWA (E. left and F. top). Her2 bands were normalized to β-actin. The data represent means ± SD of triplicate experiments (E. right and F. bottom). * P
Figure Legend Snippet: JWA is required for degradation of the HER2 protein ( A ) The SGC-7901 and NCI-N87 cells were transfected with Flag-JWA or JWA siRNA for 48 h. Western blot showed the expressions of HER2 and JWA. ( B and C ) SGC-7901 cells were transfected with JWA siRNA for 48 h, followed by treatment with 178 μM of CHX at indicated times. The protein stability of HER2 was assessed by western blotting (B). Quantification of HER2 protein levels (C). ( D ) The SGC-7901 cells were transfected with JWA siRNA, His-Ub and HER2-WT for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. The NCI-N87 cells were transfected with Flag-JWA and His-Ub for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. Western blotting was performed to confirm the levels of HER2 and JWA. Immunoprecipitation was used to show the ubiquitination of HER2. ( E and F ) SGC-7901 cells transfected with JWA siRNA or NCI-N87 cells transfected with Flag-JWA plasmid for 24 h, then incubated with or without leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of HER2 and JWA (E. left and F. top). Her2 bands were normalized to β-actin. The data represent means ± SD of triplicate experiments (E. right and F. bottom). * P

Techniques Used: Transfection, Western Blot, Immunoprecipitation, Plasmid Preparation, Incubation

Related Articles

MTT Assay:

Article Title: Colistin-Induced Nephrotoxicity in Mice Involves the Mitochondrial, Death Receptor, and Endoplasmic Reticulum Pathways
Article Snippet: .. Sodium dodecyl sulfonate (SDS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were obtained from AMRESCO Inc. (Solon, OH, USA). .. All animal studies were approved by the Institutional Animal Care and Use Committee at the China Agricultural University.

Protease Inhibitor:

Article Title: The role of Rak in the regulation of stability and function of BRCA1
Article Snippet: .. Western blot analysis, immunoprecipitation and immunofluorescence Cells were harvested, washed and lysed in modified RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonyl phenoxypolyethoxylethanol (NP-40), 0.25% sodium deoxycholate, 1 mM sodium fluoride (NaF) 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin with Complete protease inhibitor cocktail (Amresco, Solon, OH) for 1 h at 4°C. .. Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane.

Immunoprecipitation:

Article Title: The role of Rak in the regulation of stability and function of BRCA1
Article Snippet: .. Western blot analysis, immunoprecipitation and immunofluorescence Cells were harvested, washed and lysed in modified RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonyl phenoxypolyethoxylethanol (NP-40), 0.25% sodium deoxycholate, 1 mM sodium fluoride (NaF) 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin with Complete protease inhibitor cocktail (Amresco, Solon, OH) for 1 h at 4°C. .. Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane.

Incubation:

Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas
Article Snippet: .. To examine internalized antibodies and the trafficking to lysosomes, the cells were incubated with 5 μg·mL−1 anti‐CD30‐LDP at 37 °C for 24 h in the presence of 50 μg·mL−1 leupeptin (Amresco, Solon, OH, USA) and 25 μg·mL−1 pepstatin (Amresco) to inhibit lysosomal degradation. ..

other:

Article Title: Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence
Article Snippet: Leupeptin was from Amresco (J580).

Lysis:

Article Title: MiR-27b-3p Inhibition Enhances Browning of Epididymal Fat in High-Fat Diet Induced Obese Mice
Article Snippet: .. Total protein was extracted with radioimmunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% [v/v] Nonidet P-40, 1 mM EDTA, 1 mM NaF, 10 μg/mL aprotinin, 10 μM leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF); Amresco, OH, USA) and allowed to stand on ice for 30 min to permit lysis. ..

Article Title: The role of Rak in the regulation of stability and function of BRCA1
Article Snippet: .. Western blot analysis, immunoprecipitation and immunofluorescence Cells were harvested, washed and lysed in modified RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonyl phenoxypolyethoxylethanol (NP-40), 0.25% sodium deoxycholate, 1 mM sodium fluoride (NaF) 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin with Complete protease inhibitor cocktail (Amresco, Solon, OH) for 1 h at 4°C. .. Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane.

Modification:

Article Title: The role of Rak in the regulation of stability and function of BRCA1
Article Snippet: .. Western blot analysis, immunoprecipitation and immunofluorescence Cells were harvested, washed and lysed in modified RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonyl phenoxypolyethoxylethanol (NP-40), 0.25% sodium deoxycholate, 1 mM sodium fluoride (NaF) 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin with Complete protease inhibitor cocktail (Amresco, Solon, OH) for 1 h at 4°C. .. Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane.

Western Blot:

Article Title: The role of Rak in the regulation of stability and function of BRCA1
Article Snippet: .. Western blot analysis, immunoprecipitation and immunofluorescence Cells were harvested, washed and lysed in modified RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonyl phenoxypolyethoxylethanol (NP-40), 0.25% sodium deoxycholate, 1 mM sodium fluoride (NaF) 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin with Complete protease inhibitor cocktail (Amresco, Solon, OH) for 1 h at 4°C. .. Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane.

Immunofluorescence:

Article Title: The role of Rak in the regulation of stability and function of BRCA1
Article Snippet: .. Western blot analysis, immunoprecipitation and immunofluorescence Cells were harvested, washed and lysed in modified RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonyl phenoxypolyethoxylethanol (NP-40), 0.25% sodium deoxycholate, 1 mM sodium fluoride (NaF) 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin with Complete protease inhibitor cocktail (Amresco, Solon, OH) for 1 h at 4°C. .. Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane.

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    Amresco leupeptin
    Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or <t>leupeptin</t> (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p
    Leupeptin, supplied by Amresco, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptin/product/Amresco
    Average 93 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    leupeptin - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

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    Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or leupeptin (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p

    Journal: Autophagy

    Article Title: Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

    doi: 10.1080/15548627.2016.1247143

    Figure Lengend Snippet: Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or leupeptin (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p

    Article Snippet: Leupeptin was from Amresco (J580).

    Techniques: Fluorescence, Staining, Expressing, Quantitative RT-PCR, Transfection, shRNA, Western Blot, Cell Culture, Incubation

    Glucose deprivation differentially affects c-MYC protein stability in HeLa and MDA-MB-231 cells. ( a ) Western blot detection of c-MYC in HeLa and MDA-MB-231 cells treated with CHX (0.1 mM) and MG-132 (10 μ M) in the presence or absence of 25 mM glucose for 12 h. ( b ) HEK293T cells cotransfected with c-MYC-Flag and HA-Ub were treated with MG-132 in the presence or absence of glucose for 12 h. Ubiquitination of c-MYC was determined. ( c ) Western blot detection of c-MYC in HeLa cells treated with Bafilomycin A1 (Baf) (500 nM), Leupeptin (10 μ g/ml) or 3-MA (1 mM) in the presence or absence of glucose for 12 h. ( d ) HeLa cells were treated with CHX for the indicated time in the presence or absence of glucose. Representative immunoblots of c-MYC in three independent experiments were shown. Bottom, quantification of the c-MYC levels was shown. ( e and f ) HeLa ( e ) and MDA-MB-231 ( f ) stable cells ectopically expressing c-MYC-Flag were treated with MG-132 in the presence or absence of glucose. Exogenous c-MYC stability was examined. ( g ) Western blot detection of c-MYC in HeLa stable cells expressing WT HA-SKP2 or HA-tagged dominant negative mutant HA-SKP2(ΔF-box) under GD condition for 12 h. ( h ) HEK293T cells were transfected with WT-c-MYC-Flag or c-MYC(T58A)-Flag. Cell lysates were subjected to immunoprecipitation and phosphorylation of c-MYC was examined. ( i ) Western blot detection of c-MYC in HeLa stable cells expressing FBXW7 treated as in g .

    Journal: Cell Death Discovery

    Article Title: c-MYC responds to glucose deprivation in a cell-type-dependent manner

    doi: 10.1038/cddiscovery.2015.57

    Figure Lengend Snippet: Glucose deprivation differentially affects c-MYC protein stability in HeLa and MDA-MB-231 cells. ( a ) Western blot detection of c-MYC in HeLa and MDA-MB-231 cells treated with CHX (0.1 mM) and MG-132 (10 μ M) in the presence or absence of 25 mM glucose for 12 h. ( b ) HEK293T cells cotransfected with c-MYC-Flag and HA-Ub were treated with MG-132 in the presence or absence of glucose for 12 h. Ubiquitination of c-MYC was determined. ( c ) Western blot detection of c-MYC in HeLa cells treated with Bafilomycin A1 (Baf) (500 nM), Leupeptin (10 μ g/ml) or 3-MA (1 mM) in the presence or absence of glucose for 12 h. ( d ) HeLa cells were treated with CHX for the indicated time in the presence or absence of glucose. Representative immunoblots of c-MYC in three independent experiments were shown. Bottom, quantification of the c-MYC levels was shown. ( e and f ) HeLa ( e ) and MDA-MB-231 ( f ) stable cells ectopically expressing c-MYC-Flag were treated with MG-132 in the presence or absence of glucose. Exogenous c-MYC stability was examined. ( g ) Western blot detection of c-MYC in HeLa stable cells expressing WT HA-SKP2 or HA-tagged dominant negative mutant HA-SKP2(ΔF-box) under GD condition for 12 h. ( h ) HEK293T cells were transfected with WT-c-MYC-Flag or c-MYC(T58A)-Flag. Cell lysates were subjected to immunoprecipitation and phosphorylation of c-MYC was examined. ( i ) Western blot detection of c-MYC in HeLa stable cells expressing FBXW7 treated as in g .

    Article Snippet: Leupeptin was purchased from Amresco (Solon, OH, USA).

    Techniques: Multiple Displacement Amplification, Western Blot, Expressing, Dominant Negative Mutation, Transfection, Immunoprecipitation

    JWA is required for degradation of the HER2 protein ( A ) The SGC-7901 and NCI-N87 cells were transfected with Flag-JWA or JWA siRNA for 48 h. Western blot showed the expressions of HER2 and JWA. ( B and C ) SGC-7901 cells were transfected with JWA siRNA for 48 h, followed by treatment with 178 μM of CHX at indicated times. The protein stability of HER2 was assessed by western blotting (B). Quantification of HER2 protein levels (C). ( D ) The SGC-7901 cells were transfected with JWA siRNA, His-Ub and HER2-WT for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. The NCI-N87 cells were transfected with Flag-JWA and His-Ub for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. Western blotting was performed to confirm the levels of HER2 and JWA. Immunoprecipitation was used to show the ubiquitination of HER2. ( E and F ) SGC-7901 cells transfected with JWA siRNA or NCI-N87 cells transfected with Flag-JWA plasmid for 24 h, then incubated with or without leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of HER2 and JWA (E. left and F. top). Her2 bands were normalized to β-actin. The data represent means ± SD of triplicate experiments (E. right and F. bottom). * P

    Journal: Oncotarget

    Article Title: JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells

    doi: 10.18632/oncotarget.12374

    Figure Lengend Snippet: JWA is required for degradation of the HER2 protein ( A ) The SGC-7901 and NCI-N87 cells were transfected with Flag-JWA or JWA siRNA for 48 h. Western blot showed the expressions of HER2 and JWA. ( B and C ) SGC-7901 cells were transfected with JWA siRNA for 48 h, followed by treatment with 178 μM of CHX at indicated times. The protein stability of HER2 was assessed by western blotting (B). Quantification of HER2 protein levels (C). ( D ) The SGC-7901 cells were transfected with JWA siRNA, His-Ub and HER2-WT for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. The NCI-N87 cells were transfected with Flag-JWA and His-Ub for 48 h, followed by treatment with 50 μM of PS-341 for 6 h. Western blotting was performed to confirm the levels of HER2 and JWA. Immunoprecipitation was used to show the ubiquitination of HER2. ( E and F ) SGC-7901 cells transfected with JWA siRNA or NCI-N87 cells transfected with Flag-JWA plasmid for 24 h, then incubated with or without leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of HER2 and JWA (E. left and F. top). Her2 bands were normalized to β-actin. The data represent means ± SD of triplicate experiments (E. right and F. bottom). * P

    Article Snippet: Leupeptin was bought from Amresco (USA).

    Techniques: Transfection, Western Blot, Immunoprecipitation, Plasmid Preparation, Incubation