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Beyotime leupeptin
Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or <t>leupeptin</t> (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p
Leupeptin, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells"

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

Journal: Scientific Reports

doi: 10.1038/srep11009

Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p
Figure Legend Snippet: Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p

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

Related Articles

Protein Concentration:

Article Title: MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1
Article Snippet: .. Western blotting Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). .. Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C.

Radio Immunoprecipitation:

Article Title: Erythropoietin protects lipopolysaccharide-induced renal mesangial cells from autophagy
Article Snippet: .. Western blot analysis A total of 0.1 ml pre-chilled radioimmunoprecipitation assay buffer [50 mmol/l Tris-Cl (pH 7.6), 150 mmol/l NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 1 μl/ml leupeptin, 1 μl/ml aprotinin and 0.5 mmol/l phenylmethylsulfonyl fluoride] (Beyotime Institute of Biotechnology) was added to the homogenate and chilled on ice for 30 min. .. The sample was then centrifuged at 15,000 × g for 30 min, the supernatant was pipetted and the concentration of protein was measured with a bicinchoninic acid protein assay kit (Baiwang Biotechnology Corporation, Shenzhen, China).

Protease Inhibitor:

Article Title: MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1
Article Snippet: .. Western blotting Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). .. Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C.

Bicinchoninic Acid Protein Assay:

Article Title: MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1
Article Snippet: .. Western blotting Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). .. Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C.

Western Blot:

Article Title: Lentivirus-based RNA Silencing of Nemo-like Kinase (NLK) Inhibits the CAL 27 Human Adenosquamos Carcinoma Cells Proliferation and Blocks G0/G1 Phase to S Phase
Article Snippet: .. Western blot After infecting with lentivirus, CAL-27 cells were collected and lysed with RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3 VO4 , 1 µg/ml leupeptin) with PMSF (Beyotime Biotechnology, Jiangsu, China). .. Protein samples were then separated by SDS-PAGE and transferred to the PVDF membranes.

Article Title: Erythropoietin protects lipopolysaccharide-induced renal mesangial cells from autophagy
Article Snippet: .. Western blot analysis A total of 0.1 ml pre-chilled radioimmunoprecipitation assay buffer [50 mmol/l Tris-Cl (pH 7.6), 150 mmol/l NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 1 μl/ml leupeptin, 1 μl/ml aprotinin and 0.5 mmol/l phenylmethylsulfonyl fluoride] (Beyotime Institute of Biotechnology) was added to the homogenate and chilled on ice for 30 min. .. The sample was then centrifuged at 15,000 × g for 30 min, the supernatant was pipetted and the concentration of protein was measured with a bicinchoninic acid protein assay kit (Baiwang Biotechnology Corporation, Shenzhen, China).

Article Title: Novel 1,4-naphthoquinone derivatives induce reactive oxygen species-mediated apoptosis in liver cancer cells
Article Snippet: .. Western blot analysis Harvested Hep3B cells were lysed in lysis buffer (50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mg/ml AEBSF, 0.5 mg/ml pepstatin, 0.5 mg/ml leupeptin and 2 mg/ml aprotinin; Beyotime Institute of Biotechnology), The protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), following which protein lysates (30 µg) were resolved on 8–12% SDS-PAGE and electrotransferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). .. The membranes were blocked for 2 h at room temperature in fresh 5% non-fat milk in 10 mM Tris-HCl containing 150 mM NaCl (TBS; pH 7.5) and TBS+0.2% Tween-20 (TBST), followed by incubation with specific primary antibodies (all obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) against mouse monoclonal α-tubulin (1:2,500; cat. no. sc-8035), Bcl-2 (1:1,500; cat. no. sc-7382), Bax (1:1,500; cat. no. sc-493), cleaved (cle)-poly (adenosine 5-diphosphate-ribose) polymerase (cle-PARP; 1:1,500; cat. no. sc-8007), cle-caspase-3 (1:1,500; cat. no. sc-373730), phosphorylated (p)-p38 (Tyr182 , 1:1,500; cat. no. sc-7973), p-JNK (Tyr183 and Tyr185 , 1:1,500; cat. no. sc-6254), JNK (1:1,500; cat. no. sc-7345), p-ERK (Tyr204 , 1:1,500; cat. no. sc-8059), p-STAT3 (Tyr705 , 1:1,500; cat. no. sc-8059) and STAT3 (1:1,500; cat. no. sc-8019).

Article Title: MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1
Article Snippet: .. Western blotting Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). .. Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C.

Infection:

Article Title: A novel contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting autophagy in host cells
Article Snippet: .. Infected cells were collected and lysed in RIPA buffer (50 mM Tris.HCl, 150 mM NaCl, 1% (w v−1 ) NP-40, 5% (w v−1 ) sodium deoxycholate, 0.1% (w v−1 ) SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 2 μg ml−1 leupeptin) on ice for 10 min. Total protein of cell lysates was measured using BCA assay kit (Beyotime) and 45 μg protein from each sample was loaded into each lane. .. Proteins were separated by 12% SDS-PAGE, followed by transfer onto a nitrocellulose membrane (Pall Corporation).

BIA-KA:

Article Title: A novel contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting autophagy in host cells
Article Snippet: .. Infected cells were collected and lysed in RIPA buffer (50 mM Tris.HCl, 150 mM NaCl, 1% (w v−1 ) NP-40, 5% (w v−1 ) sodium deoxycholate, 0.1% (w v−1 ) SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 2 μg ml−1 leupeptin) on ice for 10 min. Total protein of cell lysates was measured using BCA assay kit (Beyotime) and 45 μg protein from each sample was loaded into each lane. .. Proteins were separated by 12% SDS-PAGE, followed by transfer onto a nitrocellulose membrane (Pall Corporation).

Article Title: MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1
Article Snippet: .. Western blotting Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). .. Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C.

Lysis:

Article Title: Sevoflurane postconditioning reduces myocardial reperfusion injury in rat isolated hearts via activation of PI3K/Akt signaling and modulation of Bcl-2 family proteins *
Article Snippet: .. The lysis buffer contained 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% (v/v) Triton X-100, 2.5 mmol/L sodium pyrophosphate, β-glycerophosphate, 1 mmol/L Na3 VO4 , 1 mmol/L ethylenediaminetetraacetic acid (EDTA), leupeptin, 1 mmol/L phenylmethanesulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology, China), and 1 mmol/L complete proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA). ..

Article Title: Novel 1,4-naphthoquinone derivatives induce reactive oxygen species-mediated apoptosis in liver cancer cells
Article Snippet: .. Western blot analysis Harvested Hep3B cells were lysed in lysis buffer (50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mg/ml AEBSF, 0.5 mg/ml pepstatin, 0.5 mg/ml leupeptin and 2 mg/ml aprotinin; Beyotime Institute of Biotechnology), The protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), following which protein lysates (30 µg) were resolved on 8–12% SDS-PAGE and electrotransferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). .. The membranes were blocked for 2 h at room temperature in fresh 5% non-fat milk in 10 mM Tris-HCl containing 150 mM NaCl (TBS; pH 7.5) and TBS+0.2% Tween-20 (TBST), followed by incubation with specific primary antibodies (all obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) against mouse monoclonal α-tubulin (1:2,500; cat. no. sc-8035), Bcl-2 (1:1,500; cat. no. sc-7382), Bax (1:1,500; cat. no. sc-493), cleaved (cle)-poly (adenosine 5-diphosphate-ribose) polymerase (cle-PARP; 1:1,500; cat. no. sc-8007), cle-caspase-3 (1:1,500; cat. no. sc-373730), phosphorylated (p)-p38 (Tyr182 , 1:1,500; cat. no. sc-7973), p-JNK (Tyr183 and Tyr185 , 1:1,500; cat. no. sc-6254), JNK (1:1,500; cat. no. sc-7345), p-ERK (Tyr204 , 1:1,500; cat. no. sc-8059), p-STAT3 (Tyr705 , 1:1,500; cat. no. sc-8059) and STAT3 (1:1,500; cat. no. sc-8019).

Article Title: Luteolin sensitizes the antitumor effect of cisplatin in drug-resistant ovarian cancer via induction of apoptosis and inhibition of cell migration and invasion
Article Snippet: .. CAOV3/DDP cells were seeded into 6-well plates (2 × 105 /well),and treated with increasing doses of luteolin (0, 10, 50, 100 μM) or cisplatin (2 μg/ml) or both for 48 h. Then, the cells were harvested, and total proteins were extracted using cell lysis buffer (1 mM PMSF, 50 mM Tris (pH 8.1), 1% SDS, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, leupeptin and other inhibitors) (Beyotime Biotechnology, Shanghai, China. ..

Article Title: MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1
Article Snippet: .. Western blotting Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). .. Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C.

SDS Page:

Article Title: Novel 1,4-naphthoquinone derivatives induce reactive oxygen species-mediated apoptosis in liver cancer cells
Article Snippet: .. Western blot analysis Harvested Hep3B cells were lysed in lysis buffer (50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mg/ml AEBSF, 0.5 mg/ml pepstatin, 0.5 mg/ml leupeptin and 2 mg/ml aprotinin; Beyotime Institute of Biotechnology), The protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), following which protein lysates (30 µg) were resolved on 8–12% SDS-PAGE and electrotransferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). .. The membranes were blocked for 2 h at room temperature in fresh 5% non-fat milk in 10 mM Tris-HCl containing 150 mM NaCl (TBS; pH 7.5) and TBS+0.2% Tween-20 (TBST), followed by incubation with specific primary antibodies (all obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) against mouse monoclonal α-tubulin (1:2,500; cat. no. sc-8035), Bcl-2 (1:1,500; cat. no. sc-7382), Bax (1:1,500; cat. no. sc-493), cleaved (cle)-poly (adenosine 5-diphosphate-ribose) polymerase (cle-PARP; 1:1,500; cat. no. sc-8007), cle-caspase-3 (1:1,500; cat. no. sc-373730), phosphorylated (p)-p38 (Tyr182 , 1:1,500; cat. no. sc-7973), p-JNK (Tyr183 and Tyr185 , 1:1,500; cat. no. sc-6254), JNK (1:1,500; cat. no. sc-7345), p-ERK (Tyr204 , 1:1,500; cat. no. sc-8059), p-STAT3 (Tyr705 , 1:1,500; cat. no. sc-8059) and STAT3 (1:1,500; cat. no. sc-8019).

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    Beyotime leupeptin
    Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or <t>leupeptin</t> (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p
    Leupeptin, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptin/product/Beyotime
    Average 94 stars, based on 69 article reviews
    Price from $9.99 to $1999.99
    leupeptin - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

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    Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p

    Journal: Scientific Reports

    Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

    doi: 10.1038/srep11009

    Figure Lengend Snippet: Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p

    Article Snippet: Nocodazole, MG132 and Leupeptin were obtained from Beyotime Institute of Biotechnology (Shanghai, China).

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