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Amresco lysis buffer
Lysis Buffer, supplied by Amresco, used in various techniques. Bioz Stars score: 94/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/lysis buffer/product/Amresco
Average 94 stars, based on 124 article reviews
Price from $9.99 to $1999.99
lysis buffer - by Bioz Stars, 2020-09
94/100 stars

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Amplification:

Article Title: Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells
Article Snippet: .. For the oocytes, we added 2 μl of lysis buffer (20 IU/μl RNase inhibitor (Amresco), 0.2% Triton X-100 (Amresco)) to the tube and proceeded with polyA+ whole transcriptome amplification using the SMART-seq2 protocol [ , ] for cDNA amplification. .. The samples were subjected to 16 cycles of PCR amplification.

Multiple Displacement Amplification:

Article Title: Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods
Article Snippet: .. BT20, MDA-MB-231, or HUVEC cells were lysed using lysis buffer (Amresco, Solon, OH, USA) supplemented with 0.1% protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). ..

Protease Inhibitor:

Article Title: Targeting TRPV1 for Body Weight Control using TRPV1−/− Mice and Electroacupuncture
Article Snippet: .. Total proteins were prepared by homogenized sample in lysis buffer containing 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 0.02% NaN3 and 1 × protease inhibitor cocktail (AMRESCO). .. The extracted proteins (30 μg per sample assessed by BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane.

Article Title: A Lateral Flow Rapid Test for Human Toxocariasis Developed Using Three Toxocara canis Recombinant Antigens
Article Snippet: .. The cells were centrifuged (10,000 × g for 15 minutes at 4°C), and the pellet was collected and resuspended in ice-cold lysis buffer (50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0), containing cOmplete™ Mini ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) (one tablet per 10 mL lysis buffer) and lysozyme (Amresco, Solon, OH) at 0.5 mg/mL. .. The cell suspension was incubated (4°C for 30 minutes) on a low speed rotator and then homogenized using a French press (Glen Mills Inc., Clifton, NJ) at a pressure of 1,700 psi.

Article Title: Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods
Article Snippet: .. BT20, MDA-MB-231, or HUVEC cells were lysed using lysis buffer (Amresco, Solon, OH, USA) supplemented with 0.1% protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). ..

Article Title: Microglial AGE-Albumin Is Critical in Promoting Alcohol-Induced Neurodegeneration in Rats and Humans
Article Snippet: .. The beads were washed in a lysis buffer (1 M Tris (pH 7.5, amresco), 5 M NaCl (Sigma aldrich), 10% NP-40 (Fluka), 10% deoxycholate and protease inhibitor cocktail (Roche)), and the immunoprecipitates were resuspended in 1×SDS sample buffer. .. Total protein concentration was measured by the QUBIT method according to the manufacturer's method.

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells
Article Snippet: .. Following harvest, adherent cells were washed with cold PBS twice and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% NP-40) containing protease inhibitor cocktail (Amresco, LLC, Solon, OH, USA). .. Tissues were dissociated using the Brain Tumor Dissociation kit (cat. no. 130-095-942; Miltenyi Biotec, Inc., Auburn, CA, USA).

Article Title: Tango1 spatially organizes ER exit sites to control ER export
Article Snippet: .. Immunoprecipitation Larval fat body was dissected in PBS from 100–200 larvae and homogenized with a motorized pellet pestle in 200 µl ice-cold lysis buffer containing 10 mM Tris-HCl, pH 7.5 (cat#0497-500G; Amresco), 150 mM NaCl (cat#x190; Amresco), 0.5 mM EDTA (cat#60-00-4; Xilonghuagong), 0.5% NP-40 (cat#ZC01468; Loogene), and protease inhibitor (cat#M221-1ML; Amresco). ..

Article Title: EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus
Article Snippet: .. For pulldown followed by Western blot analysis, cells were lysed with 2 ml of lysis buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 25 mM HEPES, pH 7.3, with addition of protease inhibitor cocktail [Amresco]) per ml of wet cell pellet. .. The lysate was clarified by centrifugation (21,100 × g , 20 min) and reacted with gH-FcStrep/gL-Flag complexes that were precoupled to Strep-Tactin XT (IBA) beads.

Immunoprecipitation:

Article Title: Tango1 spatially organizes ER exit sites to control ER export
Article Snippet: .. Immunoprecipitation Larval fat body was dissected in PBS from 100–200 larvae and homogenized with a motorized pellet pestle in 200 µl ice-cold lysis buffer containing 10 mM Tris-HCl, pH 7.5 (cat#0497-500G; Amresco), 150 mM NaCl (cat#x190; Amresco), 0.5 mM EDTA (cat#60-00-4; Xilonghuagong), 0.5% NP-40 (cat#ZC01468; Loogene), and protease inhibitor (cat#M221-1ML; Amresco). ..

Western Blot:

Article Title: EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus
Article Snippet: .. For pulldown followed by Western blot analysis, cells were lysed with 2 ml of lysis buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 25 mM HEPES, pH 7.3, with addition of protease inhibitor cocktail [Amresco]) per ml of wet cell pellet. .. The lysate was clarified by centrifugation (21,100 × g , 20 min) and reacted with gH-FcStrep/gL-Flag complexes that were precoupled to Strep-Tactin XT (IBA) beads.

Lysis:

Article Title: Targeting TRPV1 for Body Weight Control using TRPV1−/− Mice and Electroacupuncture
Article Snippet: .. Total proteins were prepared by homogenized sample in lysis buffer containing 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 0.02% NaN3 and 1 × protease inhibitor cocktail (AMRESCO). .. The extracted proteins (30 μg per sample assessed by BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane.

Article Title: A Lateral Flow Rapid Test for Human Toxocariasis Developed Using Three Toxocara canis Recombinant Antigens
Article Snippet: .. The cells were centrifuged (10,000 × g for 15 minutes at 4°C), and the pellet was collected and resuspended in ice-cold lysis buffer (50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0), containing cOmplete™ Mini ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) (one tablet per 10 mL lysis buffer) and lysozyme (Amresco, Solon, OH) at 0.5 mg/mL. .. The cell suspension was incubated (4°C for 30 minutes) on a low speed rotator and then homogenized using a French press (Glen Mills Inc., Clifton, NJ) at a pressure of 1,700 psi.

Article Title: Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells
Article Snippet: .. For the oocytes, we added 2 μl of lysis buffer (20 IU/μl RNase inhibitor (Amresco), 0.2% Triton X-100 (Amresco)) to the tube and proceeded with polyA+ whole transcriptome amplification using the SMART-seq2 protocol [ , ] for cDNA amplification. .. The samples were subjected to 16 cycles of PCR amplification.

Article Title: Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods
Article Snippet: .. BT20, MDA-MB-231, or HUVEC cells were lysed using lysis buffer (Amresco, Solon, OH, USA) supplemented with 0.1% protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). ..

Article Title: Microglial AGE-Albumin Is Critical in Promoting Alcohol-Induced Neurodegeneration in Rats and Humans
Article Snippet: .. The beads were washed in a lysis buffer (1 M Tris (pH 7.5, amresco), 5 M NaCl (Sigma aldrich), 10% NP-40 (Fluka), 10% deoxycholate and protease inhibitor cocktail (Roche)), and the immunoprecipitates were resuspended in 1×SDS sample buffer. .. Total protein concentration was measured by the QUBIT method according to the manufacturer's method.

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells
Article Snippet: .. Following harvest, adherent cells were washed with cold PBS twice and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% NP-40) containing protease inhibitor cocktail (Amresco, LLC, Solon, OH, USA). .. Tissues were dissociated using the Brain Tumor Dissociation kit (cat. no. 130-095-942; Miltenyi Biotec, Inc., Auburn, CA, USA).

Article Title: Tango1 spatially organizes ER exit sites to control ER export
Article Snippet: .. Immunoprecipitation Larval fat body was dissected in PBS from 100–200 larvae and homogenized with a motorized pellet pestle in 200 µl ice-cold lysis buffer containing 10 mM Tris-HCl, pH 7.5 (cat#0497-500G; Amresco), 150 mM NaCl (cat#x190; Amresco), 0.5 mM EDTA (cat#60-00-4; Xilonghuagong), 0.5% NP-40 (cat#ZC01468; Loogene), and protease inhibitor (cat#M221-1ML; Amresco). ..

Article Title: EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus
Article Snippet: .. For pulldown followed by Western blot analysis, cells were lysed with 2 ml of lysis buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 25 mM HEPES, pH 7.3, with addition of protease inhibitor cocktail [Amresco]) per ml of wet cell pellet. .. The lysate was clarified by centrifugation (21,100 × g , 20 min) and reacted with gH-FcStrep/gL-Flag complexes that were precoupled to Strep-Tactin XT (IBA) beads.

Chloramphenicol Acetyltransferase Assay:

Article Title: Tango1 spatially organizes ER exit sites to control ER export
Article Snippet: .. Immunoprecipitation Larval fat body was dissected in PBS from 100–200 larvae and homogenized with a motorized pellet pestle in 200 µl ice-cold lysis buffer containing 10 mM Tris-HCl, pH 7.5 (cat0497-500G; Amresco), 150 mM NaCl (cat#x190; Amresco), 0.5 mM EDTA (cat#60-00-4; Xilonghuagong), 0.5% NP-40 (cat#ZC01468; Loogene), and protease inhibitor (cat#M221-1ML; Amresco). ..

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  • 90
    Amresco immunoprecipitation lysis buffer
    SIRT6 promotes the ubiquitination of p27 ( A ) 293 cells were transfected with SIRT6 and vector, twenty four hours after transfection, cells were treated with MG132 or acetyl-leu-leu-norleucinal (ALLN) 5 h before harvesting. Proteins were analyzed by western blot. ( B ) An in vivo ubiquitination assay was performed. 293 cells were transiently transfected with SIRT6 and vector, siRNA-SIRT6 and NC (negative control). Forty two hours later, cells were treated with MG132 for 5 h. Cells were lysed and proceeded for <t>co-immunoprecipitation</t> using the p27 antibody.
    Immunoprecipitation Lysis Buffer, supplied by Amresco, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoprecipitation lysis buffer/product/Amresco
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    immunoprecipitation lysis buffer - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    92
    Amresco cytosolic lysis buffer
    SUMO-2 is more efficient than SUMO-1 in regulating nuclear p38 and p-p38 during H. pylori infection. The cytoplasmic and nuclear fractions of p38 and p-p38 were analyzed from siSUMO transfectants ( A ) and RFP-SUMO transfectants ( B ). p38 and p-p38 were blotted with anti-p38 and anti-p-p38 antibodies. SUMO-1 and SUMO-2 were detected using anti-SUMO-1 and anti-SUMO-2 antibodies. RFP alone or fusion proteins of RFP-SUMO-1 or RFP-SUMO-2, were detected using anti-RFP. GAPDH and Lamin A/C were used as <t>cytosolic</t> and nuclear markers respectively. For α-SUMO-1 and α-SUMO-2 the upper panels show conjugated SUMOs while the lower panels show free-form SUMOs. ( A ) Western Blots showed that the endogenous SUMO-1 and SUMO-2 were down-regulated after transfectional incubation of siSUMO-1 and siSUMO-2. p38 and p-p38 in the N-fraction were decreased in siSUMO-1 and siSUMO-2 transfectants without H. pylori infection; however, their nuclear levels decreased only for siSUMO-2 and not for siSUMO-1 transfectants during H. pylori infection; and ( B ) Western Blots showed that RFP-SUMO-1 and RFP-SUMO-2 fusion proteins were up-regulated after transfectional incubation. p38 and p-p38 levels were up-regulated in the N-fraction and clearly down-regulated in the C-fraction in RFP-SUMO-2 transfectants during H. pylori infection. Quantification of the blots shown in ( A , B ) is summarized in Table S3 below. All experiments were repeated three times and representative images are shown.
    Cytosolic Lysis Buffer, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cytosolic lysis buffer/product/Amresco
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cytosolic lysis buffer - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    SIRT6 promotes the ubiquitination of p27 ( A ) 293 cells were transfected with SIRT6 and vector, twenty four hours after transfection, cells were treated with MG132 or acetyl-leu-leu-norleucinal (ALLN) 5 h before harvesting. Proteins were analyzed by western blot. ( B ) An in vivo ubiquitination assay was performed. 293 cells were transiently transfected with SIRT6 and vector, siRNA-SIRT6 and NC (negative control). Forty two hours later, cells were treated with MG132 for 5 h. Cells were lysed and proceeded for co-immunoprecipitation using the p27 antibody.

    Journal: Aging (Albany NY)

    Article Title: SIRT6 delays cellular senescence by promoting p27Kip1 ubiquitin-proteasome degradation

    doi: 10.18632/aging.101038

    Figure Lengend Snippet: SIRT6 promotes the ubiquitination of p27 ( A ) 293 cells were transfected with SIRT6 and vector, twenty four hours after transfection, cells were treated with MG132 or acetyl-leu-leu-norleucinal (ALLN) 5 h before harvesting. Proteins were analyzed by western blot. ( B ) An in vivo ubiquitination assay was performed. 293 cells were transiently transfected with SIRT6 and vector, siRNA-SIRT6 and NC (negative control). Forty two hours later, cells were treated with MG132 for 5 h. Cells were lysed and proceeded for co-immunoprecipitation using the p27 antibody.

    Article Snippet: Co-immunoprecipitation assays Cells for immunoprecipitation assay were lysed in immunoprecipitation lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5% glycerol) containing Protease Inhibitor Mixture (Amresco).

    Techniques: Transfection, Plasmid Preparation, Western Blot, In Vivo, Ubiquitin Assay, Negative Control, Immunoprecipitation

    PIASxα and PTEN interact with each other both in vivo and in vitro . A , co-immunoprecipitation of PIASxα with PTEN from HeLa cells. Cells were transfected with FLAG-PIASxα. 48 h after transfection, cells were harvested and lysed.

    Journal: The Journal of Biological Chemistry

    Article Title: PIASxα Ligase Enhances SUMO1 Modification of PTEN Protein as a SUMO E3 Ligase *

    doi: 10.1074/jbc.M113.508515

    Figure Lengend Snippet: PIASxα and PTEN interact with each other both in vivo and in vitro . A , co-immunoprecipitation of PIASxα with PTEN from HeLa cells. Cells were transfected with FLAG-PIASxα. 48 h after transfection, cells were harvested and lysed.

    Article Snippet: Cells for immunoprecipitation assay were lysed in immunoprecipitation lysis buffer (25 m m Tris-HCl pH 7.4, 150 m m NaCl, 1% Nonidet P-40, 1 m m EDTA, 5% glycerol) containing Protease Inhibitor Mixture (Amresco).

    Techniques: In Vivo, In Vitro, Immunoprecipitation, Transfection

    SUMO-2 is more efficient than SUMO-1 in regulating nuclear p38 and p-p38 during H. pylori infection. The cytoplasmic and nuclear fractions of p38 and p-p38 were analyzed from siSUMO transfectants ( A ) and RFP-SUMO transfectants ( B ). p38 and p-p38 were blotted with anti-p38 and anti-p-p38 antibodies. SUMO-1 and SUMO-2 were detected using anti-SUMO-1 and anti-SUMO-2 antibodies. RFP alone or fusion proteins of RFP-SUMO-1 or RFP-SUMO-2, were detected using anti-RFP. GAPDH and Lamin A/C were used as cytosolic and nuclear markers respectively. For α-SUMO-1 and α-SUMO-2 the upper panels show conjugated SUMOs while the lower panels show free-form SUMOs. ( A ) Western Blots showed that the endogenous SUMO-1 and SUMO-2 were down-regulated after transfectional incubation of siSUMO-1 and siSUMO-2. p38 and p-p38 in the N-fraction were decreased in siSUMO-1 and siSUMO-2 transfectants without H. pylori infection; however, their nuclear levels decreased only for siSUMO-2 and not for siSUMO-1 transfectants during H. pylori infection; and ( B ) Western Blots showed that RFP-SUMO-1 and RFP-SUMO-2 fusion proteins were up-regulated after transfectional incubation. p38 and p-p38 levels were up-regulated in the N-fraction and clearly down-regulated in the C-fraction in RFP-SUMO-2 transfectants during H. pylori infection. Quantification of the blots shown in ( A , B ) is summarized in Table S3 below. All experiments were repeated three times and representative images are shown.

    Journal: International Journal of Molecular Sciences

    Article Title: SUMOs Mediate the Nuclear Transfer of p38 and p-p38 during Helicobacter Pylori Infection

    doi: 10.3390/ijms19092482

    Figure Lengend Snippet: SUMO-2 is more efficient than SUMO-1 in regulating nuclear p38 and p-p38 during H. pylori infection. The cytoplasmic and nuclear fractions of p38 and p-p38 were analyzed from siSUMO transfectants ( A ) and RFP-SUMO transfectants ( B ). p38 and p-p38 were blotted with anti-p38 and anti-p-p38 antibodies. SUMO-1 and SUMO-2 were detected using anti-SUMO-1 and anti-SUMO-2 antibodies. RFP alone or fusion proteins of RFP-SUMO-1 or RFP-SUMO-2, were detected using anti-RFP. GAPDH and Lamin A/C were used as cytosolic and nuclear markers respectively. For α-SUMO-1 and α-SUMO-2 the upper panels show conjugated SUMOs while the lower panels show free-form SUMOs. ( A ) Western Blots showed that the endogenous SUMO-1 and SUMO-2 were down-regulated after transfectional incubation of siSUMO-1 and siSUMO-2. p38 and p-p38 in the N-fraction were decreased in siSUMO-1 and siSUMO-2 transfectants without H. pylori infection; however, their nuclear levels decreased only for siSUMO-2 and not for siSUMO-1 transfectants during H. pylori infection; and ( B ) Western Blots showed that RFP-SUMO-1 and RFP-SUMO-2 fusion proteins were up-regulated after transfectional incubation. p38 and p-p38 levels were up-regulated in the N-fraction and clearly down-regulated in the C-fraction in RFP-SUMO-2 transfectants during H. pylori infection. Quantification of the blots shown in ( A , B ) is summarized in Table S3 below. All experiments were repeated three times and representative images are shown.

    Article Snippet: Nuclear and Cytosolic Isolation AGS cells were harvested and resuspended in cytosolic lysis buffer (0.1% NP40 (Amresco), 10 mM Tris (pH7.9), 10 mM MgCl2 (Sigma-Aldrich), 15 mM NaCl, 50 mM NEM, and 1% protease inhibitor cocktail) on ice for 10 min. After brief centrifugation, the supernatant was saved as the cytosolic fraction, and the nuclear pellet was lysed in SDS sample buffer.

    Techniques: Infection, Western Blot, Incubation

    SUMO-2 is more efficient than SUMO-1 in regulating nuclear p38 and p-p38 during H. pylori infection. The cytoplasmic and nuclear fractions of p38 and p-p38 were analyzed from siSUMO transfectants ( A ) and RFP-SUMO transfectants ( B ). p38 and p-p38 were blotted with anti-p38 and anti-p-p38 antibodies. SUMO-1 and SUMO-2 were detected using anti-SUMO-1 and anti-SUMO-2 antibodies. RFP alone or fusion proteins of RFP-SUMO-1 or RFP-SUMO-2, were detected using anti-RFP. GAPDH and Lamin A/C were used as cytosolic and nuclear markers respectively. For α-SUMO-1 and α-SUMO-2 the upper panels show conjugated SUMOs while the lower panels show free-form SUMOs. ( A ) Western Blots showed that the endogenous SUMO-1 and SUMO-2 were down-regulated after transfectional incubation of siSUMO-1 and siSUMO-2. p38 and p-p38 in the N-fraction were decreased in siSUMO-1 and siSUMO-2 transfectants without H. pylori infection; however, their nuclear levels decreased only for siSUMO-2 and not for siSUMO-1 transfectants during H. pylori infection; and ( B ) Western Blots showed that RFP-SUMO-1 and RFP-SUMO-2 fusion proteins were up-regulated after transfectional incubation. p38 and p-p38 levels were up-regulated in the N-fraction and clearly down-regulated in the C-fraction in RFP-SUMO-2 transfectants during H. pylori infection. Quantification of the blots shown in ( A , B below. All experiments were repeated three times and representative images are shown.

    Journal: International Journal of Molecular Sciences

    Article Title: SUMOs Mediate the Nuclear Transfer of p38 and p-p38 during Helicobacter Pylori Infection

    doi: 10.3390/ijms19092482

    Figure Lengend Snippet: SUMO-2 is more efficient than SUMO-1 in regulating nuclear p38 and p-p38 during H. pylori infection. The cytoplasmic and nuclear fractions of p38 and p-p38 were analyzed from siSUMO transfectants ( A ) and RFP-SUMO transfectants ( B ). p38 and p-p38 were blotted with anti-p38 and anti-p-p38 antibodies. SUMO-1 and SUMO-2 were detected using anti-SUMO-1 and anti-SUMO-2 antibodies. RFP alone or fusion proteins of RFP-SUMO-1 or RFP-SUMO-2, were detected using anti-RFP. GAPDH and Lamin A/C were used as cytosolic and nuclear markers respectively. For α-SUMO-1 and α-SUMO-2 the upper panels show conjugated SUMOs while the lower panels show free-form SUMOs. ( A ) Western Blots showed that the endogenous SUMO-1 and SUMO-2 were down-regulated after transfectional incubation of siSUMO-1 and siSUMO-2. p38 and p-p38 in the N-fraction were decreased in siSUMO-1 and siSUMO-2 transfectants without H. pylori infection; however, their nuclear levels decreased only for siSUMO-2 and not for siSUMO-1 transfectants during H. pylori infection; and ( B ) Western Blots showed that RFP-SUMO-1 and RFP-SUMO-2 fusion proteins were up-regulated after transfectional incubation. p38 and p-p38 levels were up-regulated in the N-fraction and clearly down-regulated in the C-fraction in RFP-SUMO-2 transfectants during H. pylori infection. Quantification of the blots shown in ( A , B below. All experiments were repeated three times and representative images are shown.

    Article Snippet: AGS cells were harvested and resuspended in cytosolic lysis buffer (0.1% NP40 (Amresco), 10 mM Tris (pH7.9), 10 mM MgCl2 (Sigma-Aldrich), 15 mM NaCl, 50 mM NEM, and 1% protease inhibitor cocktail) on ice for 10 min. After brief centrifugation, the supernatant was saved as the cytosolic fraction, and the nuclear pellet was lysed in SDS sample buffer.

    Techniques: Infection, Western Blot, Incubation