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Roche np 40 lysis buffer
Isolation of HBV NCs from MNase-treated lysates by linear sucrose gradient centrifugation and detection of NC-associated viral DNA, RNA, and capsid protein. Induced HepAD38 cells were lysed by using <t>NP-40,</t> and the cytoplasmic lysate was treated with MNase.
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1) Product Images from "Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid"

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

Journal: Journal of Virology

doi: 10.1128/JVI.01912-13

Isolation of HBV NCs from MNase-treated lysates by linear sucrose gradient centrifugation and detection of NC-associated viral DNA, RNA, and capsid protein. Induced HepAD38 cells were lysed by using NP-40, and the cytoplasmic lysate was treated with MNase.
Figure Legend Snippet: Isolation of HBV NCs from MNase-treated lysates by linear sucrose gradient centrifugation and detection of NC-associated viral DNA, RNA, and capsid protein. Induced HepAD38 cells were lysed by using NP-40, and the cytoplasmic lysate was treated with MNase.

Techniques Used: Isolation, Gradient Centrifugation

2) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

Journal: Journal of Virology

doi: 10.1128/JVI.00798-18

CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.
Figure Legend Snippet: CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

Techniques Used: Gradient Centrifugation, Southern Blot, Cell Culture, Incubation, Concentration Assay

3) Product Images from "Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿"

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00321-10

Suppression of E2F1 reduces levels of productive infection. (A) RS cells were transfected with pcDNA3.1 empty vector, 100 nM E2F1 siRNA, or 100 nM control siRNA. Twenty-four hours later, cells were transfected with 1 μg of BoHV-1 gCBlue DNA. Twenty-four hours after transfection, cells were fixed and β-Gal + cells were counted. The results constitute the averages for four independent experiments. (B) RS cells were transfected with 100 nM E2F1 siRNA or 100 nM control siRNA. Forty-eight hours after transfection, cells were collected and lysed with NP-40 lysis buffer, and 100 μg of protein was electrophoresed by 12% SDS-PAGE. Proteins in the gel were transferred to a polyvinylidene difluoride membrane and probed with E2F1 antiserum diluted 1:10,000. As a control, 100 μg cell lysate was probed with antiserum that specifically recognized E2F2 or β-actin.
Figure Legend Snippet: Suppression of E2F1 reduces levels of productive infection. (A) RS cells were transfected with pcDNA3.1 empty vector, 100 nM E2F1 siRNA, or 100 nM control siRNA. Twenty-four hours later, cells were transfected with 1 μg of BoHV-1 gCBlue DNA. Twenty-four hours after transfection, cells were fixed and β-Gal + cells were counted. The results constitute the averages for four independent experiments. (B) RS cells were transfected with 100 nM E2F1 siRNA or 100 nM control siRNA. Forty-eight hours after transfection, cells were collected and lysed with NP-40 lysis buffer, and 100 μg of protein was electrophoresed by 12% SDS-PAGE. Proteins in the gel were transferred to a polyvinylidene difluoride membrane and probed with E2F1 antiserum diluted 1:10,000. As a control, 100 μg cell lysate was probed with antiserum that specifically recognized E2F2 or β-actin.

Techniques Used: Infection, Transfection, Plasmid Preparation, Lysis, SDS Page

4) Product Images from "Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿"

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00508-10

Impα3 interacts with HIV-1 integrase (IN) in vitro and in cotransfected 293T cells and HIV-infected C8166 T cells. (A) (Left) Cell lysates from 293T cells expressing GFP or GFP-IN were incubated with either GST alone or a recombinant GST-Impα3 fusion protein. After GST pulldown, the bound proteins were analyzed by Western blotting (WB) with an anti-GFP antibody. (Right) Equal amounts of GST and GST-Impα3 were incubated with purified recombinant HIV-1 IN followed by GST pulldown and were analyzed on an SDS-PAGE gel by Western blotting with rabbit anti-IN antibodies. (B) Impα3 interacts with HIV-1 IN and Vpr in 293T cells. A full-length human T7-Impα3 expression plasmid was cotransfected with a plasmid expressing GFP, GFP-IN, MA-YFP, or Vpr-YFP into 293T cells. (Top) After 48 h of transfection, 90% of the transfected cells were lysed in 0.25% NP-40 buffer and were immunoprecipitated with a rabbit anti-GFP antibody. The immunoprecipitates were resolved using 10% SDS-PAGE, and the bound T7-Impα3 was detected by WB with an anti-T7 antibody. (Center) The presence of GFP-INwt/mut in immunoprecipitates was detected by an anti-GFP antibody. (Bottom) To check protein expression, the remaining 10% of the transfected cells were lysed with 0.5% NP-40, run directly on a 10% SDS-PAGE gel, and probed with anti-T7 antibodies. (C) HeLa cells were transfected with a GFP, GFP-IN, MA-YFP, or Vpr-YFP plasmid. After 48 h, cells were fixed and labeled with an anti-GFP antibody, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were then analyzed by fluorescence microscopy (with a 40× objective lens). (D) Impα3 interacts with HIV-1 IN in virus-infected C8166 T cells. Approximately 10 × 10 6 C8166 T cells were infected with an HxBru or HxBru-IN-HA virus. (Top) Seventy-two hours after infection, cells were lysed and immunoprecipitated with a rabbit anti-HA antibody, and the bound endogenous Impα3 was detected by Western blotting using rabbit anti-Impα3. The normal C8166 cell lysate was loaded as a positive control (PC). (Center) The immunoprecipitated HA-IN was also detected by WB using a mouse anti-HA antibody. (Bottom) Lysates from the infected C8166 cells were directly loaded onto an SDS-PAGE gel to detect HIV p24 gag protein by WB using an anti-p24 antibody.
Figure Legend Snippet: Impα3 interacts with HIV-1 integrase (IN) in vitro and in cotransfected 293T cells and HIV-infected C8166 T cells. (A) (Left) Cell lysates from 293T cells expressing GFP or GFP-IN were incubated with either GST alone or a recombinant GST-Impα3 fusion protein. After GST pulldown, the bound proteins were analyzed by Western blotting (WB) with an anti-GFP antibody. (Right) Equal amounts of GST and GST-Impα3 were incubated with purified recombinant HIV-1 IN followed by GST pulldown and were analyzed on an SDS-PAGE gel by Western blotting with rabbit anti-IN antibodies. (B) Impα3 interacts with HIV-1 IN and Vpr in 293T cells. A full-length human T7-Impα3 expression plasmid was cotransfected with a plasmid expressing GFP, GFP-IN, MA-YFP, or Vpr-YFP into 293T cells. (Top) After 48 h of transfection, 90% of the transfected cells were lysed in 0.25% NP-40 buffer and were immunoprecipitated with a rabbit anti-GFP antibody. The immunoprecipitates were resolved using 10% SDS-PAGE, and the bound T7-Impα3 was detected by WB with an anti-T7 antibody. (Center) The presence of GFP-INwt/mut in immunoprecipitates was detected by an anti-GFP antibody. (Bottom) To check protein expression, the remaining 10% of the transfected cells were lysed with 0.5% NP-40, run directly on a 10% SDS-PAGE gel, and probed with anti-T7 antibodies. (C) HeLa cells were transfected with a GFP, GFP-IN, MA-YFP, or Vpr-YFP plasmid. After 48 h, cells were fixed and labeled with an anti-GFP antibody, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were then analyzed by fluorescence microscopy (with a 40× objective lens). (D) Impα3 interacts with HIV-1 IN in virus-infected C8166 T cells. Approximately 10 × 10 6 C8166 T cells were infected with an HxBru or HxBru-IN-HA virus. (Top) Seventy-two hours after infection, cells were lysed and immunoprecipitated with a rabbit anti-HA antibody, and the bound endogenous Impα3 was detected by Western blotting using rabbit anti-Impα3. The normal C8166 cell lysate was loaded as a positive control (PC). (Center) The immunoprecipitated HA-IN was also detected by WB using a mouse anti-HA antibody. (Bottom) Lysates from the infected C8166 cells were directly loaded onto an SDS-PAGE gel to detect HIV p24 gag protein by WB using an anti-p24 antibody.

Techniques Used: In Vitro, Infection, Expressing, Incubation, Recombinant, Western Blot, Purification, SDS Page, Plasmid Preparation, Transfection, Immunoprecipitation, Labeling, Staining, Fluorescence, Microscopy, Positive Control

5) Product Images from "Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy"

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2019.00053

Central fragment of Reelin (R3–6) does not induce Dab1 phosphorylation. HEK293 cells expressing Dab1_myc and ApoER2 (A) or Dab1_myc and VLDLR (B) or Dab1_myc, ApoER2, and VLDLR (C) were kept in Imaging Medium for 30 min. Next, the medium was changed to RCM containing full length Reelin (FL Rln, lanes 2) or R3–6 (lanes 3) was added to the cells (final concentration, 30 nM) or cells were left untreated (neg.ctrl, lanes 1). After 20 min incubation at 37°C, cells were washed and lysed in NP-40 lysis buffer. Lysates were analyzed by western blotting using phospho-Dab1 antibody (Ab pDab1) and GAPDH antibody. Membranes were stripped and subsequently re-probed for the detection of Dab1 (AbD4). ApoER2 was detected by Ab20 and VLDLR by AbAF2258.
Figure Legend Snippet: Central fragment of Reelin (R3–6) does not induce Dab1 phosphorylation. HEK293 cells expressing Dab1_myc and ApoER2 (A) or Dab1_myc and VLDLR (B) or Dab1_myc, ApoER2, and VLDLR (C) were kept in Imaging Medium for 30 min. Next, the medium was changed to RCM containing full length Reelin (FL Rln, lanes 2) or R3–6 (lanes 3) was added to the cells (final concentration, 30 nM) or cells were left untreated (neg.ctrl, lanes 1). After 20 min incubation at 37°C, cells were washed and lysed in NP-40 lysis buffer. Lysates were analyzed by western blotting using phospho-Dab1 antibody (Ab pDab1) and GAPDH antibody. Membranes were stripped and subsequently re-probed for the detection of Dab1 (AbD4). ApoER2 was detected by Ab20 and VLDLR by AbAF2258.

Techniques Used: Expressing, Imaging, Concentration Assay, Incubation, Lysis, Western Blot

6) Product Images from "PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma"

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma

Journal: Oncotarget

doi: 10.18632/oncotarget.20864

Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.
Figure Legend Snippet: Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.

Techniques Used: Activation Assay, Inhibition, Western Blot, Lysis, Immunoprecipitation

7) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

Journal: Journal of Virology

doi: 10.1128/JVI.00798-18

CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.
Figure Legend Snippet: CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

Techniques Used: Gradient Centrifugation, Southern Blot, Cell Culture, Incubation, Concentration Assay

8) Product Images from "Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins"

Article Title: Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins

Journal: Journal of Virology

doi: 10.1128/JVI.76.12.6037-6043.2002

Triton X-114 phase separation of surface proteins from ST cells. The proteins recovered from the aqueous (a) and detergent (d) phases were electrophoretically separated and either visualized by silver staining (right panel) or, following transfer to nitrocellulose, analyzed for binding to the S protein of TGEV (left panel). The positions of molecular mass markers are indicated, as are the locations of aminopeptidase N and the high-molecular-mass sialoglycoprotein recognized by S (arrows).
Figure Legend Snippet: Triton X-114 phase separation of surface proteins from ST cells. The proteins recovered from the aqueous (a) and detergent (d) phases were electrophoretically separated and either visualized by silver staining (right panel) or, following transfer to nitrocellulose, analyzed for binding to the S protein of TGEV (left panel). The positions of molecular mass markers are indicated, as are the locations of aminopeptidase N and the high-molecular-mass sialoglycoprotein recognized by S (arrows).

Techniques Used: Silver Staining, Binding Assay

9) Product Images from "Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It"

Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024434

mGBP2 and mGBP5 interact with mGBP1 and accumulate on type II T. gondii (Pru) vacuoles. (A) mGBP2, mGBP4, and mGBP5 were identified as interactors of mGBP1 in a immunoprecipitation with subsequent MS/MS analysis (A). The left panel shows a silver-stained SDS-PAGE gel following an anti-FLAG IP from wild-type RAW264.7 cells (control lane) or RAW264.7 that overexpress wild-type or various mGBP1 mutants. The right panel shows three GBPs that were identified in the mass spectrometry analysis as binding partners of mGBP1. Wild-type RAW264.7 cells or RAW264.7 cells that overexpress FLAG-mGBP1, FLAG-mGBP1(R48A) or FLAG-mGBP1(K51A) were stimulated with 200 U/ml IFN-γ overnight and infected with type II T. gondii at an MOI of 5–10 for 1 h. The mGBP1 protein was recovered from all lanes except from the control lane (*). Unique peptides recovered from mGBP2, mGBP4, and mGBP5 are shown in red and peptides common with mGBP1 are shown in blue. The total number of unique amino acids (# AA), the percentage of total mass (% Mass), and the percentage of total amino acids (% AA) recovered are also shown. For mGBP4 and mGBP5, the sequence coverage was 3–4%. 14% of the mGBP2-specific amino acids were found in the gel (21% when including amino acids common to mGBP1). (B) Interaction of HA-mGBP2 and HA-mGBP5 with FLAG-mGBP1 was confirmed by co-IP with subsequent immunoblot analysis. RAW264.7 cells that overexpress either FLAG-mGBP1 and HA-mGBP2, FLAG-mGBP1 and HA-mGBP5, or FLAG-mGBP1 alone were lysed in 0.5% NP-40 and proteins were immunoprecipitated with an anti-HA antibody. Co-IP of FLAG-mGBP1 was confirmed by immunoblot with an anti-FLAG antibody. A mouse monoclonal anti-FLAG antibody (Sigma-Aldrich) and a rat monoclonal anti-HA antibody (Roche Applied Science) were used. (C) mGBP2 and mGBP5 are recruited to type II T. gondii vacuoles in an IFN-γ dependent manner. Confocal pictures show MEFs that overexpress either eGFP-mGBP2 or eGFP-mGBP5 pre-induced with 200 U/ml IFN-γ and infected with type II T. gondii . Live imaging was performed with a spinning disk confocal microscope and revealed that both mGBP2 and mGBP5 were targeted to T. gondii vacuoles in an IFN-γ-dependent manner.
Figure Legend Snippet: mGBP2 and mGBP5 interact with mGBP1 and accumulate on type II T. gondii (Pru) vacuoles. (A) mGBP2, mGBP4, and mGBP5 were identified as interactors of mGBP1 in a immunoprecipitation with subsequent MS/MS analysis (A). The left panel shows a silver-stained SDS-PAGE gel following an anti-FLAG IP from wild-type RAW264.7 cells (control lane) or RAW264.7 that overexpress wild-type or various mGBP1 mutants. The right panel shows three GBPs that were identified in the mass spectrometry analysis as binding partners of mGBP1. Wild-type RAW264.7 cells or RAW264.7 cells that overexpress FLAG-mGBP1, FLAG-mGBP1(R48A) or FLAG-mGBP1(K51A) were stimulated with 200 U/ml IFN-γ overnight and infected with type II T. gondii at an MOI of 5–10 for 1 h. The mGBP1 protein was recovered from all lanes except from the control lane (*). Unique peptides recovered from mGBP2, mGBP4, and mGBP5 are shown in red and peptides common with mGBP1 are shown in blue. The total number of unique amino acids (# AA), the percentage of total mass (% Mass), and the percentage of total amino acids (% AA) recovered are also shown. For mGBP4 and mGBP5, the sequence coverage was 3–4%. 14% of the mGBP2-specific amino acids were found in the gel (21% when including amino acids common to mGBP1). (B) Interaction of HA-mGBP2 and HA-mGBP5 with FLAG-mGBP1 was confirmed by co-IP with subsequent immunoblot analysis. RAW264.7 cells that overexpress either FLAG-mGBP1 and HA-mGBP2, FLAG-mGBP1 and HA-mGBP5, or FLAG-mGBP1 alone were lysed in 0.5% NP-40 and proteins were immunoprecipitated with an anti-HA antibody. Co-IP of FLAG-mGBP1 was confirmed by immunoblot with an anti-FLAG antibody. A mouse monoclonal anti-FLAG antibody (Sigma-Aldrich) and a rat monoclonal anti-HA antibody (Roche Applied Science) were used. (C) mGBP2 and mGBP5 are recruited to type II T. gondii vacuoles in an IFN-γ dependent manner. Confocal pictures show MEFs that overexpress either eGFP-mGBP2 or eGFP-mGBP5 pre-induced with 200 U/ml IFN-γ and infected with type II T. gondii . Live imaging was performed with a spinning disk confocal microscope and revealed that both mGBP2 and mGBP5 were targeted to T. gondii vacuoles in an IFN-γ-dependent manner.

Techniques Used: Immunoprecipitation, Mass Spectrometry, Staining, SDS Page, Binding Assay, Infection, Sequencing, Co-Immunoprecipitation Assay, Imaging, Microscopy

10) Product Images from "4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion"

Article Title: 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008143

4-BPB tags a large number of proteins by click chemistry. Parasites were treated with either DMSO, 4-PPB or 4-APB and then lysed. The supernatants were then treated with the alkyne- or azide-conjugated biotin-tags, and click chemistry was used to label the targets of the 4-BPB click derivatives. These targets were then purified using streptavidin-conjugated beads and resolved by SDS-PAGE and silver staining. The resulting bands in the silver-stained gel were cut out for identification by LC-MS/MS. Parasites were either treated with DMSO, 0.5 µM 4-PPB or 1 µM 4-APB for 10 minutes, followed by lysis in 1% NP-40 buffer. Each of the lysates was treated with either the 4-APB-tag or the 4-PPB-tag during the click reaction. Lanes are labeled with the inhibitor (DMSO (lanes 1,2), 4-PPB (lanes 3,4), 4-APB (lanes 5,6)) and the click tag (4PPB-tag (lanes 1,3,5) or 4-APB-tag (lanes 2,4,6)) that the corresponding sample was treated with.
Figure Legend Snippet: 4-BPB tags a large number of proteins by click chemistry. Parasites were treated with either DMSO, 4-PPB or 4-APB and then lysed. The supernatants were then treated with the alkyne- or azide-conjugated biotin-tags, and click chemistry was used to label the targets of the 4-BPB click derivatives. These targets were then purified using streptavidin-conjugated beads and resolved by SDS-PAGE and silver staining. The resulting bands in the silver-stained gel were cut out for identification by LC-MS/MS. Parasites were either treated with DMSO, 0.5 µM 4-PPB or 1 µM 4-APB for 10 minutes, followed by lysis in 1% NP-40 buffer. Each of the lysates was treated with either the 4-APB-tag or the 4-PPB-tag during the click reaction. Lanes are labeled with the inhibitor (DMSO (lanes 1,2), 4-PPB (lanes 3,4), 4-APB (lanes 5,6)) and the click tag (4PPB-tag (lanes 1,3,5) or 4-APB-tag (lanes 2,4,6)) that the corresponding sample was treated with.

Techniques Used: Purification, SDS Page, Silver Staining, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Lysis, Labeling

11) Product Images from "VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E"

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E

Journal: Virology

doi: 10.1016/j.virol.2010.06.007

Coimmunoprecipitation of gE and VP16 with VP22 truncation mutants (A) VP22 truncation mutants fused to GFP. A schematic representation of full-length and N-terminal truncation mutants of VP22 fused to the N-terminus of the GFP protein. Also represented, deletion of the region of VP22 that facilitates binding to both VP16 and gE and this domain alone fused to GFP. (B) Expression of VP22 mutants in transfected/infected cells. Vero cells were transfected with the VP22-GFP constructs represented in Fig. 6A, and 20 h later, they were infected with a VP22-null virus [U L 49 − ]. After an additional 10-h incubation, the cells were lysed with NP-40 lysis buffer. A fraction of each cell lysate was analyzed by Western blotting using a goat polyclonal antibody specific for GFP. The remainder of the transfected/infected cell lysate was incubated with a goat polyclonal antibody against GFP and resulting antibody-antigen complexes were collected with protein G-agarose beads. After extensive washes with lysis buffer, material that immunoprecipitated with anti-GFP antibody was separated by SDS-PAGE and transferred to nitrocellulose. Coimmunoprecipitated gE (C) and VP16 (D) were detected by immunoblot using a rabbit polyclonal antibody specific for gE or a rabbit monospecific polyclonal antibody raised against a C-terminal peptide of VP16. The positions of molecular mass markers (in kilodaltons) are indicated on the left.
Figure Legend Snippet: Coimmunoprecipitation of gE and VP16 with VP22 truncation mutants (A) VP22 truncation mutants fused to GFP. A schematic representation of full-length and N-terminal truncation mutants of VP22 fused to the N-terminus of the GFP protein. Also represented, deletion of the region of VP22 that facilitates binding to both VP16 and gE and this domain alone fused to GFP. (B) Expression of VP22 mutants in transfected/infected cells. Vero cells were transfected with the VP22-GFP constructs represented in Fig. 6A, and 20 h later, they were infected with a VP22-null virus [U L 49 − ]. After an additional 10-h incubation, the cells were lysed with NP-40 lysis buffer. A fraction of each cell lysate was analyzed by Western blotting using a goat polyclonal antibody specific for GFP. The remainder of the transfected/infected cell lysate was incubated with a goat polyclonal antibody against GFP and resulting antibody-antigen complexes were collected with protein G-agarose beads. After extensive washes with lysis buffer, material that immunoprecipitated with anti-GFP antibody was separated by SDS-PAGE and transferred to nitrocellulose. Coimmunoprecipitated gE (C) and VP16 (D) were detected by immunoblot using a rabbit polyclonal antibody specific for gE or a rabbit monospecific polyclonal antibody raised against a C-terminal peptide of VP16. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

Techniques Used: Binding Assay, Expressing, Transfection, Infection, Construct, Incubation, Lysis, Western Blot, Immunoprecipitation, SDS Page

Characterization of the ability of VP22 point mutants to bind to the cytoplasmic tail of gE in a GST pulldown assay (A) Point mutants of VP22 fused to GFP. Schematic representation of wild-type VP22 and amino acid substitution mutants used in this study fused to the N-terminus of the GFP protein. (B) and (C) Expression of VP22 point mutants in transfected cells. Vero cells were transfected with the indicated constructs and 20 h post-transfection, the monolayers were lysed with NP-40 lysis buffer. Expression of VP22-GFP fusion proteins was analyzed by Western blotting using a rabbit polyclonal antibody specific for GFP. (D) and (E) GST pulldown from transfected cell lysates using GST-gECT. Approximately equal amounts of purified GST fusion proteins were added to the remainder of the transfected-cell lysates. Beads were washed extensively with lysis buffer and bound proteins were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit polyclonal antibody raised against the GFP protein. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (F) Binding efficiency of VP22 point mutants to the cytoplasmic tail of gE. Using densitometry, binding efficiency was quantitated by dividing the amount of VP22-GFP protein detected in the pulldown assay (normalized for the amount of GST-gECT present) by the amount in the cell lysate (normalized for the amount of actin present). In each experiment, the wild-type VP22-GFP construct was set at 100% binding efficiency. Error bars represent standard deviations for four replicate experiments.
Figure Legend Snippet: Characterization of the ability of VP22 point mutants to bind to the cytoplasmic tail of gE in a GST pulldown assay (A) Point mutants of VP22 fused to GFP. Schematic representation of wild-type VP22 and amino acid substitution mutants used in this study fused to the N-terminus of the GFP protein. (B) and (C) Expression of VP22 point mutants in transfected cells. Vero cells were transfected with the indicated constructs and 20 h post-transfection, the monolayers were lysed with NP-40 lysis buffer. Expression of VP22-GFP fusion proteins was analyzed by Western blotting using a rabbit polyclonal antibody specific for GFP. (D) and (E) GST pulldown from transfected cell lysates using GST-gECT. Approximately equal amounts of purified GST fusion proteins were added to the remainder of the transfected-cell lysates. Beads were washed extensively with lysis buffer and bound proteins were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit polyclonal antibody raised against the GFP protein. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (F) Binding efficiency of VP22 point mutants to the cytoplasmic tail of gE. Using densitometry, binding efficiency was quantitated by dividing the amount of VP22-GFP protein detected in the pulldown assay (normalized for the amount of GST-gECT present) by the amount in the cell lysate (normalized for the amount of actin present). In each experiment, the wild-type VP22-GFP construct was set at 100% binding efficiency. Error bars represent standard deviations for four replicate experiments.

Techniques Used: GST Pulldown Assay, Expressing, Transfection, Construct, Lysis, Western Blot, Purification, SDS Page, Binding Assay

12) Product Images from "The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿"

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01856-10

Na interacts with TRAF2. 293 cells were cotransfected with the indicated combinations of vector control, FLAG-Na, or HA-TRAF2. Cell lysates were harvested in NP-40 buffer 48 h posttransfection. Immunoprecipitation analysis was performed using antibodies
Figure Legend Snippet: Na interacts with TRAF2. 293 cells were cotransfected with the indicated combinations of vector control, FLAG-Na, or HA-TRAF2. Cell lysates were harvested in NP-40 buffer 48 h posttransfection. Immunoprecipitation analysis was performed using antibodies

Techniques Used: Plasmid Preparation, Immunoprecipitation

13) Product Images from "The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome"

Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

Journal: Nature Communications

doi: 10.1038/s41467-018-03669-z

Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p
Figure Legend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

Techniques Used: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

14) Product Images from "LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State"

Article Title: LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2017.00118

APP dimer generation and processing takes place in primary cortical neurons. (A) Murine primary cortical neurons (DIV 7) were infected with an adenoviral vector encoding human APP695 while (B) HEK 293T cells were transiently transfected with the pLHCX-APP695 wt construct. 24 h post infection or transfection, respectively, conditioned media (CM) were collected and cells were lysed in RIPA (PCN) or NP-40 (HEK) lysis buffer. Via the antibody mix 1G75A3 (1:3,000) APP was detected in lysates (upper blots) and conditioned media (lower blots). PCN show similar APP dimer expression in the lysate as HEK cells and also generate soluble APP dimers. Under reducing conditions using β-mercaptoethanol (βME) and heating at 95°C the dimer band disappeared. All lanes of lysate or conditioned medium are on the same blot but were rearranged for better presentation.
Figure Legend Snippet: APP dimer generation and processing takes place in primary cortical neurons. (A) Murine primary cortical neurons (DIV 7) were infected with an adenoviral vector encoding human APP695 while (B) HEK 293T cells were transiently transfected with the pLHCX-APP695 wt construct. 24 h post infection or transfection, respectively, conditioned media (CM) were collected and cells were lysed in RIPA (PCN) or NP-40 (HEK) lysis buffer. Via the antibody mix 1G75A3 (1:3,000) APP was detected in lysates (upper blots) and conditioned media (lower blots). PCN show similar APP dimer expression in the lysate as HEK cells and also generate soluble APP dimers. Under reducing conditions using β-mercaptoethanol (βME) and heating at 95°C the dimer band disappeared. All lanes of lysate or conditioned medium are on the same blot but were rearranged for better presentation.

Techniques Used: Infection, Plasmid Preparation, Transfection, Construct, Lysis, Expressing

15) Product Images from "Structural and functional analysis of the GABARAP interaction motif (GIM)"

Article Title: Structural and functional analysis of the GABARAP interaction motif (GIM)

Journal: EMBO Reports

doi: 10.15252/embr.201643587

PLEKHM1 preferentially interacts with GABARAP in vitro and in vivo ITC titrations of PLEKHM1‐LIR peptide into LC3 family proteins (top panel) and GABARAP family proteins (bottom panel). The top diagrams in each ITC plot display the raw measurements, and the bottom diagrams show the integrated heat per titration step. Best fit is presented as a solid line. GFP‐tagged LC3/GABARAP proteins were expressed alone or with PLEKHM1‐WT‐Flag in HEK293T cells and immunoprecipitated using GFP‐Trap beads and blotted for the presence or absence of PLEKHM1 (anti‐Flag tag). Free GFP was observed in lanes three to six (GFP‐LC3A and GFP‐LC3B) potentially due to lysosomal turnover. GFP‐LC3/GABARAPs were overexpressed in HeLa cells and treated for 4 h with KU‐0063794 (10 μM) plus chloroquine (20 μM), immunoprecipitated with GFP‐Trap beads and blotted for the presence of endogenous PLEKHM1. Plekhm1 +/+ or Plekhm1 −/− mouse embryonic fibroblasts were either treated with vehicle (DMSO) or treated for 4 h with KU‐0063794 (10 μM) plus chloroquine (20 μM). Samples were lysed in NP‐40 lysis buffer, and endogenous PLEKHM1 was immunoprecipitated in the presence of 50 μM PLEKHM1‐LIR peptide (KVRPQQ EDEWVNV QYPDQPE) or 50 μM Scrambled (Scr) PLEKHM1‐LIR peptide (VQEQQEPPPVKNYDVEQWDR). Samples were then immunoblotted for the presence of endogenous PLEKHM1, LC3B and GABARAP proteins. Source data are available online for this figure.
Figure Legend Snippet: PLEKHM1 preferentially interacts with GABARAP in vitro and in vivo ITC titrations of PLEKHM1‐LIR peptide into LC3 family proteins (top panel) and GABARAP family proteins (bottom panel). The top diagrams in each ITC plot display the raw measurements, and the bottom diagrams show the integrated heat per titration step. Best fit is presented as a solid line. GFP‐tagged LC3/GABARAP proteins were expressed alone or with PLEKHM1‐WT‐Flag in HEK293T cells and immunoprecipitated using GFP‐Trap beads and blotted for the presence or absence of PLEKHM1 (anti‐Flag tag). Free GFP was observed in lanes three to six (GFP‐LC3A and GFP‐LC3B) potentially due to lysosomal turnover. GFP‐LC3/GABARAPs were overexpressed in HeLa cells and treated for 4 h with KU‐0063794 (10 μM) plus chloroquine (20 μM), immunoprecipitated with GFP‐Trap beads and blotted for the presence of endogenous PLEKHM1. Plekhm1 +/+ or Plekhm1 −/− mouse embryonic fibroblasts were either treated with vehicle (DMSO) or treated for 4 h with KU‐0063794 (10 μM) plus chloroquine (20 μM). Samples were lysed in NP‐40 lysis buffer, and endogenous PLEKHM1 was immunoprecipitated in the presence of 50 μM PLEKHM1‐LIR peptide (KVRPQQ EDEWVNV QYPDQPE) or 50 μM Scrambled (Scr) PLEKHM1‐LIR peptide (VQEQQEPPPVKNYDVEQWDR). Samples were then immunoblotted for the presence of endogenous PLEKHM1, LC3B and GABARAP proteins. Source data are available online for this figure.

Techniques Used: In Vitro, In Vivo, Titration, Immunoprecipitation, FLAG-tag, Lysis

16) Product Images from "Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver"

Article Title: Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-009-0120-2

MAb OC.10 reacts with protein immunoprecipitated with anti-Hsc70 antibody. BDE4 cell extract (500 µg) prepared in NP-40 lysis buffer with 2 mM ADP was immunoprecipitated with 5 µg of rat monoclonal anti-Hsc70 (
Figure Legend Snippet: MAb OC.10 reacts with protein immunoprecipitated with anti-Hsc70 antibody. BDE4 cell extract (500 µg) prepared in NP-40 lysis buffer with 2 mM ADP was immunoprecipitated with 5 µg of rat monoclonal anti-Hsc70 (

Techniques Used: Immunoprecipitation, Lysis

17) Product Images from "An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication"

Article Title: An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003147

The L uAUG modulates translation of the L pORF in response to eIF2α phosphorylation in 293T cells. A . Diagram of the pCAGGS EBOV L 5′-UTR firefly luciferase fusion reporter construct with the EBOV L 5′-UTR upstream of L (amino acids 1–13) in frame with firefly luciferase (L-FF) and an identical construct, lacking the uAUG codon (Lns-FF). B . Thapsigargin (TG) treatment induces eIF2α∼P. Cells were treated with either DMSO or with increasing doses of TG and lysed in NP-40 lysis buffer with protease inhibitors. eIF2α∼P levels were measured by western blot. C . Western blot of total eIF2α and eIF2α∼P levels, from untreated and TG-treated cells which were lysed in passive lysis buffer that was used for the luciferase analysis in panel D. D . The L uAUG functions to maintain L translation following TG treatment. 293T cells were transfected with pRLTK and the L-FF or the Lns-FF reporter constructs. At 24 hpt, cells were treated with four doses of TG and harvested at 10 hours post treatment. Dual luciferase assays were performed to determine the firefly to Renilla luciferase ratio in the presence or absence of TG treatment and the FF to Renilla ratio of the DMSO treated cells transfected with L-FF was set to 1. Each data point represents the mean and standard deviation of four replicates.
Figure Legend Snippet: The L uAUG modulates translation of the L pORF in response to eIF2α phosphorylation in 293T cells. A . Diagram of the pCAGGS EBOV L 5′-UTR firefly luciferase fusion reporter construct with the EBOV L 5′-UTR upstream of L (amino acids 1–13) in frame with firefly luciferase (L-FF) and an identical construct, lacking the uAUG codon (Lns-FF). B . Thapsigargin (TG) treatment induces eIF2α∼P. Cells were treated with either DMSO or with increasing doses of TG and lysed in NP-40 lysis buffer with protease inhibitors. eIF2α∼P levels were measured by western blot. C . Western blot of total eIF2α and eIF2α∼P levels, from untreated and TG-treated cells which were lysed in passive lysis buffer that was used for the luciferase analysis in panel D. D . The L uAUG functions to maintain L translation following TG treatment. 293T cells were transfected with pRLTK and the L-FF or the Lns-FF reporter constructs. At 24 hpt, cells were treated with four doses of TG and harvested at 10 hours post treatment. Dual luciferase assays were performed to determine the firefly to Renilla luciferase ratio in the presence or absence of TG treatment and the FF to Renilla ratio of the DMSO treated cells transfected with L-FF was set to 1. Each data point represents the mean and standard deviation of four replicates.

Techniques Used: Luciferase, Construct, Lysis, Western Blot, Transfection, Standard Deviation

18) Product Images from "Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy"

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2019.00053

Central fragment of Reelin (R3–6) does not induce Dab1 phosphorylation. HEK293 cells expressing Dab1_myc and ApoER2 (A) or Dab1_myc and VLDLR (B) or Dab1_myc, ApoER2, and VLDLR (C) were kept in Imaging Medium for 30 min. Next, the medium was changed to RCM containing full length Reelin (FL Rln, lanes 2) or R3–6 (lanes 3) was added to the cells (final concentration, 30 nM) or cells were left untreated (neg.ctrl, lanes 1). After 20 min incubation at 37°C, cells were washed and lysed in NP-40 lysis buffer. Lysates were analyzed by western blotting using phospho-Dab1 antibody (Ab pDab1) and GAPDH antibody. Membranes were stripped and subsequently re-probed for the detection of Dab1 (AbD4). ApoER2 was detected by Ab20 and VLDLR by AbAF2258.
Figure Legend Snippet: Central fragment of Reelin (R3–6) does not induce Dab1 phosphorylation. HEK293 cells expressing Dab1_myc and ApoER2 (A) or Dab1_myc and VLDLR (B) or Dab1_myc, ApoER2, and VLDLR (C) were kept in Imaging Medium for 30 min. Next, the medium was changed to RCM containing full length Reelin (FL Rln, lanes 2) or R3–6 (lanes 3) was added to the cells (final concentration, 30 nM) or cells were left untreated (neg.ctrl, lanes 1). After 20 min incubation at 37°C, cells were washed and lysed in NP-40 lysis buffer. Lysates were analyzed by western blotting using phospho-Dab1 antibody (Ab pDab1) and GAPDH antibody. Membranes were stripped and subsequently re-probed for the detection of Dab1 (AbD4). ApoER2 was detected by Ab20 and VLDLR by AbAF2258.

Techniques Used: Expressing, Imaging, Concentration Assay, Incubation, Lysis, Western Blot

19) Product Images from "XBP-1-deficient plasmablasts show normal protein folding but altered glycosylation and lipid synthesis"

Article Title: XBP-1-deficient plasmablasts show normal protein folding but altered glycosylation and lipid synthesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0900953

(A)  Naïve B cells from XBP-1 WT , XBP-1 WT /MD4 and XBP-1 KO /MD4 mice were cultured in the presence of LPS for 4 days and then labeled with [ 35 S]-methionine/cysteine for 4 h. Triton X-114 lysis and separation were performed. Both the pellet and soluble
Figure Legend Snippet: (A) Naïve B cells from XBP-1 WT , XBP-1 WT /MD4 and XBP-1 KO /MD4 mice were cultured in the presence of LPS for 4 days and then labeled with [ 35 S]-methionine/cysteine for 4 h. Triton X-114 lysis and separation were performed. Both the pellet and soluble

Techniques Used: Mouse Assay, Cell Culture, Labeling, Lysis

(A)  Four-day LPS-stimulated XBP-1 WT /MD4 and XBP-1 KO /MD4 B cells were lysed in Triton X-114, and secreted and membrane IgM were separated as described. Lysates were then immunoblotted using the anti-μ antibody.  (B)  Naïve and 3-day LPS-stimulated
Figure Legend Snippet: (A) Four-day LPS-stimulated XBP-1 WT /MD4 and XBP-1 KO /MD4 B cells were lysed in Triton X-114, and secreted and membrane IgM were separated as described. Lysates were then immunoblotted using the anti-μ antibody. (B) Naïve and 3-day LPS-stimulated

Techniques Used:

20) Product Images from "The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome"

Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

Journal: Nature Communications

doi: 10.1038/s41467-018-03669-z

Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p
Figure Legend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

Techniques Used: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

21) Product Images from "Dexamethasone Treatment of Calves Latently Infected with Bovine Herpesvirus 1 Leads to Activation of the bICP0 Early Promoter, in Part by the Cellular Transcription Factor C/EBP-Alpha "

Article Title: Dexamethasone Treatment of Calves Latently Infected with Bovine Herpesvirus 1 Leads to Activation of the bICP0 Early Promoter, in Part by the Cellular Transcription Factor C/EBP-Alpha

Journal: Journal of Virology

doi: 10.1128/JVI.01009-09

C/EBP-alpha directly interacts with the bICP0 E promoter. (A) Neuro-2A cells were transfected with 1 μg of C/EBPα. At 48 h posttransfection, cells were collected and lysed with NP-40 lysis buffer. Thirty or 100 μg of protein were
Figure Legend Snippet: C/EBP-alpha directly interacts with the bICP0 E promoter. (A) Neuro-2A cells were transfected with 1 μg of C/EBPα. At 48 h posttransfection, cells were collected and lysed with NP-40 lysis buffer. Thirty or 100 μg of protein were

Techniques Used: Transfection, Lysis

22) Product Images from "Direct Measurement of Cathepsin B Activity in the Cytosol of Apoptotic Cells by an Activity-Based Probe"

Article Title: Direct Measurement of Cathepsin B Activity in the Cytosol of Apoptotic Cells by an Activity-Based Probe

Journal: Chemistry & biology

doi: 10.1016/j.chembiol.2009.07.011

Probes 1 and 3 Read Out the Release of CatB into the Cytosol during Apoptosis (A) CatB cleavage of caspase-like substrates at lysosomal and cytosolic pH. Purified CatB was incubated with several fluorogenic caspase peptide substrates for 30 min at 37°C and fluorescence of released amino-methylcoumarin (AMC; excitation at 380 nM; emission at 460 nM) was measured. Fluorescence was normalized to a canonical CatB substrate, Z-FR-AMC. (B) Measurement of CatB activity in the cytosol during the intrinsic pathway of apoptosis. NIH 3T3 cells were treated with staurosporine (1 μM) or DMSO vehicle for 3 hr. The cytosol was then extracted with digitonin (labeled cytosol) and the remaining pellet was resuspended in NP-40 lysis buffer (labeled NP40 soluble). The fluorescent substrate Z-FR-AMC was then added and fluorescence was measured. The cathepsin inhibitor E64 inhibited the fluorescence signals. (C) Competition of probe 3 labeling by the CatB inhibitor E64. NIH 3T3 cells were either pretreated with E64 (50 μM) for 1 hr prior to activation with staurosporine (1 μM) or DMSO vehicle for 3 hr. Staurosporine-treated cells were then treated with 3 alone (300 nM or 5 μM) or cotreated with 3 (300 nM or 5 mM) and E64 (50 μM) for 30 min prior to lysis and cycloaddition. Nonapoptotic cells were treated with 3 alone (5 μM) or cotreated with 3 (5 μM) and E64 (50 μM) for 30 min prior to lysis and cycloaddition. All samples were then analyzed by streptavidin blotting. (D) Comparison of staurosporine-induced caspase activity in CatB null and wild-type fibroblasts. CatB null or wild-type fibroblasts were treated with staurosporine (1 μM) and caspase activity was measured at different treatment times using a caspase-dependent luminescence kit. Experiments in (A), (B), and (D) were performed in triplicate and errors are standard deviation (*p
Figure Legend Snippet: Probes 1 and 3 Read Out the Release of CatB into the Cytosol during Apoptosis (A) CatB cleavage of caspase-like substrates at lysosomal and cytosolic pH. Purified CatB was incubated with several fluorogenic caspase peptide substrates for 30 min at 37°C and fluorescence of released amino-methylcoumarin (AMC; excitation at 380 nM; emission at 460 nM) was measured. Fluorescence was normalized to a canonical CatB substrate, Z-FR-AMC. (B) Measurement of CatB activity in the cytosol during the intrinsic pathway of apoptosis. NIH 3T3 cells were treated with staurosporine (1 μM) or DMSO vehicle for 3 hr. The cytosol was then extracted with digitonin (labeled cytosol) and the remaining pellet was resuspended in NP-40 lysis buffer (labeled NP40 soluble). The fluorescent substrate Z-FR-AMC was then added and fluorescence was measured. The cathepsin inhibitor E64 inhibited the fluorescence signals. (C) Competition of probe 3 labeling by the CatB inhibitor E64. NIH 3T3 cells were either pretreated with E64 (50 μM) for 1 hr prior to activation with staurosporine (1 μM) or DMSO vehicle for 3 hr. Staurosporine-treated cells were then treated with 3 alone (300 nM or 5 μM) or cotreated with 3 (300 nM or 5 mM) and E64 (50 μM) for 30 min prior to lysis and cycloaddition. Nonapoptotic cells were treated with 3 alone (5 μM) or cotreated with 3 (5 μM) and E64 (50 μM) for 30 min prior to lysis and cycloaddition. All samples were then analyzed by streptavidin blotting. (D) Comparison of staurosporine-induced caspase activity in CatB null and wild-type fibroblasts. CatB null or wild-type fibroblasts were treated with staurosporine (1 μM) and caspase activity was measured at different treatment times using a caspase-dependent luminescence kit. Experiments in (A), (B), and (D) were performed in triplicate and errors are standard deviation (*p

Techniques Used: Purification, Incubation, Fluorescence, Activity Assay, Labeling, Lysis, Activation Assay, Standard Deviation

23) Product Images from "A Protein Encoded by the Bovine Herpesvirus 1 Latency-Related Gene Interacts with Specific Cellular Regulatory Proteins, Including CCAAT Enhancer Binding Protein Alpha ▿"

Article Title: A Protein Encoded by the Bovine Herpesvirus 1 Latency-Related Gene Interacts with Specific Cellular Regulatory Proteins, Including CCAAT Enhancer Binding Protein Alpha ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01171-06

Expression of C/EBP-α during productive infection. MDBK cells were infected with wt BHV-1 (V) using a multiplicity of infection of 5, and whole-cell lysate was collected at 6, 14, 24, or 30 h after infection. M, mock-infected cells. Cells were lysed with NP-40 lysis buffer, and 250 μg of protein was electrophoresed by 12% SDS-PAGE. Proteins in the gel were transferred onto a PVDF membrane and probed with anti-C/EBP-α that was diluted 1:500. The arrow denotes the 42-kDa isoform of C/EBP-α, and the closed circle denotes a 35-kDa band that was recognized by the antibody. Molecular weight markers are in kilodaltons.
Figure Legend Snippet: Expression of C/EBP-α during productive infection. MDBK cells were infected with wt BHV-1 (V) using a multiplicity of infection of 5, and whole-cell lysate was collected at 6, 14, 24, or 30 h after infection. M, mock-infected cells. Cells were lysed with NP-40 lysis buffer, and 250 μg of protein was electrophoresed by 12% SDS-PAGE. Proteins in the gel were transferred onto a PVDF membrane and probed with anti-C/EBP-α that was diluted 1:500. The arrow denotes the 42-kDa isoform of C/EBP-α, and the closed circle denotes a 35-kDa band that was recognized by the antibody. Molecular weight markers are in kilodaltons.

Techniques Used: Expressing, Infection, Lysis, SDS Page, Molecular Weight

24) Product Images from "A small molecule FAK kinase inhibitor, GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells"

Article Title: A small molecule FAK kinase inhibitor, GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells

Journal: Cell Cycle

doi: 10.4161/15384101.2014.949550

GSK2256098 inhibition of Akt and ERK phosphorylation. PANC-1and L3.6P1 cells were cultured in DMEM or GMEM medium containing 10% FBS and antibiotics. Cell monolayers were lysed in NP-40 lysis buffer supplemented with protease inhibitors. Proteins were
Figure Legend Snippet: GSK2256098 inhibition of Akt and ERK phosphorylation. PANC-1and L3.6P1 cells were cultured in DMEM or GMEM medium containing 10% FBS and antibiotics. Cell monolayers were lysed in NP-40 lysis buffer supplemented with protease inhibitors. Proteins were

Techniques Used: Inhibition, Cell Culture, Lysis

The effects of GSK2256098 on PARP and caspase levels. PANC-1 and L3.6P1 cells were cultured in DMEM or GMEM medium containing 10% FBS and antibiotics. Cell monolayers were lysed in NP-40 lysis buffer supplemented with protease inhibitors. Proteins were
Figure Legend Snippet: The effects of GSK2256098 on PARP and caspase levels. PANC-1 and L3.6P1 cells were cultured in DMEM or GMEM medium containing 10% FBS and antibiotics. Cell monolayers were lysed in NP-40 lysis buffer supplemented with protease inhibitors. Proteins were

Techniques Used: Cell Culture, Lysis

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Centrifugation:

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF. .. Protein concentrations were determined by BCA protein assay (Pierce) and were equalized among samples.

Article Title: Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins
Article Snippet: .. The cells were pelleted by centrifugation (693 × g , 5 min, 4°C) and resuspended in 1 ml of NP-40 lysis buffer (1% 4-nonylphenolpolyethyleneglycol, 0.5% deoxycholate, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing protease inhibitors (protease inhibitor cocktail Complete; Roche). .. Following incubation on ice for 15 min, insoluble material was removed by centrifugation (16,000 × g , 30 min, 4°C).

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
Article Snippet: .. Co-Immunoprecipitation IFN-γ-induced RAW264.7 cells were lysed for 30 min at 4°C in 0.5% NP-40 lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and 0.5 mM GTP) and nuclear fraction was removed by centrifugation. .. Supernatants were pre-cleared for 3 h at 4°C with Protein G beads and subsequently incubated for 2 h at 4°C with a rat monoclonal anti-HA antibody (Roche Applied Science) and Protein G beads.

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: A 1 ml sample of the cell suspension was removed for analysis of expression of the VP22-GFP fusion proteins, and the remaining cells were pelleted by centrifugation (1000 × g for 5 min at 4 °C). .. The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C). .. After centrifugation, the clarified supernatant was subjected to immunoprecipitation using a rabbit anti-GFP antibody.

Autoradiography:

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer. .. Coimmunoprecipitated VP16 and gE were detected by immunoblot using a rabbit monospecific polyclonal antibody raised against a C-terminal peptide of the HSV-1 tegument protein VP16 (Clontech), or a rabbit polyclonal antibody specific for gE (a kind gift from Harvey Friedman, University of Pennsylvania, Philadelphia, Pennsylvania), a goat anti-rabbit antibody conjugated to horseradish peroxidase, ECL reagents, and chemiluminescence autoradiography on Kodak BioMax XAR film.

Construct:

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: Confluent monolayers of Vero cells grown in 100-mm plates were transfected with the indicated constructs by using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. .. The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer.

SDS-Gel:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Bound proteins were eluted with sodium dodecyl sulfate (SDS)-gel loading buffer, resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and detected by Western blotting using mouse anti-GFP antibodies. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Incubation:

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: .. To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase.

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF. .. Cell lysates were incubated with CD19 (Santa Cruz), BCAP (R & D) or IgG (goat, Santa Cruz) at 4°C.

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris. .. In the first method, the lysate was incubated with micrococcal nuclease (MNase) (Roche) (150 units/ml) and CaCl2 (5 mM) at 37°C for 90 min to degrade the nucleic acids outside NCs.

Article Title: Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins
Article Snippet: The cells were pelleted by centrifugation (693 × g , 5 min, 4°C) and resuspended in 1 ml of NP-40 lysis buffer (1% 4-nonylphenolpolyethyleneglycol, 0.5% deoxycholate, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing protease inhibitors (protease inhibitor cocktail Complete; Roche). .. Following incubation on ice for 15 min, insoluble material was removed by centrifugation (16,000 × g , 30 min, 4°C).

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: .. After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 . .. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿
Article Snippet: 293 cells were transfected with the indicated expression plasmids, and cell lysates were harvested in NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, 0.75% IPEGAL) containing protease inhibitor cocktail (Roche) 48 h posttransfection. .. Aliquots of 150 μg of protein were incubated with 1 μg FLAG or HA antibodies for 2 h. Protein-antibody complexes were then incubated with 25 μl protein A/G-agarose beads (Santa Cruz Biotechnology).

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
Article Snippet: Co-Immunoprecipitation IFN-γ-induced RAW264.7 cells were lysed for 30 min at 4°C in 0.5% NP-40 lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and 0.5 mM GTP) and nuclear fraction was removed by centrifugation. .. Supernatants were pre-cleared for 3 h at 4°C with Protein G beads and subsequently incubated for 2 h at 4°C with a rat monoclonal anti-HA antibody (Roche Applied Science) and Protein G beads.

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer. .. The lysates were then incubated with a goat polyclonal antibody raised against GFP (Rockland) for 1 h at 4 °C, and immune complexes were collected with protein G-agarose beads that had been washed twice with lysis buffer.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Then 100 μl of glutathione-Sepharose 4B beads (Amersham Biosciences) was added to the mixture and incubated for 2 h at 4°C. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Cell Culture:

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Expressing:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: HepAD38 cells expressing the HBV pgRNA under the control of a tetracycline (Tet)-repressible promoter were derived from the human hepatoblastoma cell line HepG-2 ( ) and maintained in Dulbecco modified Eagle–F-12 medium supplemented with 10% fetal bovine serum (FBS) and 5 μg/ml of Tet until induction. .. HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris.

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿
Article Snippet: .. 293 cells were transfected with the indicated expression plasmids, and cell lysates were harvested in NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, 0.75% IPEGAL) containing protease inhibitor cocktail (Roche) 48 h posttransfection. ..

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: A 1 ml sample of the cell suspension was removed for analysis of expression of the VP22-GFP fusion proteins, and the remaining cells were pelleted by centrifugation (1000 × g for 5 min at 4 °C). .. The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: To detect the interaction between Impα3 and HIV IN, Vpr, MA, or different IN deletion mutants in mammalian cells, 293T cells were cotransfected with T7-Impα3 and the AcGFP-INwt/mut, MA-YFP, or Vpr-YFP expression plasmid. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

BIA-KA:

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF. .. Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF.

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 . .. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Modification:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: HepAD38 cells expressing the HBV pgRNA under the control of a tetracycline (Tet)-repressible promoter were derived from the human hepatoblastoma cell line HepG-2 ( ) and maintained in Dulbecco modified Eagle–F-12 medium supplemented with 10% fetal bovine serum (FBS) and 5 μg/ml of Tet until induction. .. HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris.

Western Blot:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Bound proteins were eluted with sodium dodecyl sulfate (SDS)-gel loading buffer, resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and detected by Western blotting using mouse anti-GFP antibodies. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Derivative Assay:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: HepAD38 cells expressing the HBV pgRNA under the control of a tetracycline (Tet)-repressible promoter were derived from the human hepatoblastoma cell line HepG-2 ( ) and maintained in Dulbecco modified Eagle–F-12 medium supplemented with 10% fetal bovine serum (FBS) and 5 μg/ml of Tet until induction. .. HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris.

Transfection:

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: Cells were transfected with pcDNA5myc_Dab1 and pClneo_ApoER2 or pcDNA5myc_Dab1 and pClneo_VLDLR or pcDNA5myc_Dab1, pClneo_ApoER2, and pClneo_VLDLR. .. After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 .

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿
Article Snippet: .. 293 cells were transfected with the indicated expression plasmids, and cell lysates were harvested in NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, 0.75% IPEGAL) containing protease inhibitor cocktail (Roche) 48 h posttransfection. ..

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Forty-eight hours after transfection, whole-cell lysate was prepared. .. Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml).

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: Confluent monolayers of Vero cells grown in 100-mm plates were transfected with the indicated constructs by using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. .. The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer.

Concentration Assay:

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: .. To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase.

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: After 24 h, cells were washed with PBS and kept in Imaging Medium (Hank’s Balanced Salt Solution, 2 mM Glutamine, 10 mM HEPES) for 30 min. Next, medium was changed to Reelin conditioned medium (RCM) or R3–6 was added to the cells (final concentration, 30 nM). .. After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 .

Protease Inhibitor:

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF. .. Protein concentrations were determined by BCA protein assay (Pierce) and were equalized among samples.

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: .. HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris. ..

Article Title: Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins
Article Snippet: .. The cells were pelleted by centrifugation (693 × g , 5 min, 4°C) and resuspended in 1 ml of NP-40 lysis buffer (1% 4-nonylphenolpolyethyleneglycol, 0.5% deoxycholate, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing protease inhibitors (protease inhibitor cocktail Complete; Roche). .. Following incubation on ice for 15 min, insoluble material was removed by centrifugation (16,000 × g , 30 min, 4°C).

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: .. After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 . .. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿
Article Snippet: .. 293 cells were transfected with the indicated expression plasmids, and cell lysates were harvested in NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, 0.75% IPEGAL) containing protease inhibitor cocktail (Roche) 48 h posttransfection. ..

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: .. Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
Article Snippet: .. Co-Immunoprecipitation IFN-γ-induced RAW264.7 cells were lysed for 30 min at 4°C in 0.5% NP-40 lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and 0.5 mM GTP) and nuclear fraction was removed by centrifugation. .. Supernatants were pre-cleared for 3 h at 4°C with Protein G beads and subsequently incubated for 2 h at 4°C with a rat monoclonal anti-HA antibody (Roche Applied Science) and Protein G beads.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C). .. After centrifugation, the clarified supernatant was subjected to immunoprecipitation using a rabbit anti-GFP antibody.

Infection:

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: At 20 h post-transfection, the monolayers were infected with HSV-1 KOS strain or a VP22-null virus [UL 49− ] at a MOI of 10. .. The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer.

Imaging:

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: After 24 h, cells were washed with PBS and kept in Imaging Medium (Hank’s Balanced Salt Solution, 2 mM Glutamine, 10 mM HEPES) for 30 min. Next, medium was changed to Reelin conditioned medium (RCM) or R3–6 was added to the cells (final concentration, 30 nM). .. After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 .

Protein Concentration:

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 . .. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Sonication:

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Binding Assay:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Paragraph title: In vitro binding assay and cell-based coimmunoprecipitation (co-IP) experiments. ... Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Nucleic Acid Electrophoresis:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Bound proteins were eluted with sodium dodecyl sulfate (SDS)-gel loading buffer, resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and detected by Western blotting using mouse anti-GFP antibodies. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Isolation:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: Paragraph title: Isolation of HBV NCs from cell lysates and virions by sucrose gradient centrifugation. ... HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris.

Electrophoretic Mobility Shift Assay:

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: Paragraph title: Electrophoretic mobility shift assay (EMSA). ... Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml).

Purification:

Article Title: 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion
Article Snippet: Protein Labeling and Click Chemistry Parasites were purified as described above and treated either with DMSO, 4-PPB or 4-APB at different concentrations for 15 minutes at room temperature. .. Parasites were spun down and washed three times in PBS, and resuspended in 1% NP-40 lysis buffer containing Complete (Roche) protease inhibitors.

Labeling:

Article Title: 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion
Article Snippet: Paragraph title: Protein Labeling and Click Chemistry ... Parasites were spun down and washed three times in PBS, and resuspended in 1% NP-40 lysis buffer containing Complete (Roche) protease inhibitors.

Polyacrylamide Gel Electrophoresis:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Bound proteins were eluted with sodium dodecyl sulfate (SDS)-gel loading buffer, resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and detected by Western blotting using mouse anti-GFP antibodies. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

SDS Page:

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 . .. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Article Title: 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion
Article Snippet: Parasites were spun down and washed three times in PBS, and resuspended in 1% NP-40 lysis buffer containing Complete (Roche) protease inhibitors. .. Samples were then either mixed with sample buffer for SDS-PAGE and subsequent treatment with streptavidin-HRP (Sigma), or used directly for precipitation.

Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
Article Snippet: Co-Immunoprecipitation IFN-γ-induced RAW264.7 cells were lysed for 30 min at 4°C in 0.5% NP-40 lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and 0.5 mM GTP) and nuclear fraction was removed by centrifugation. .. Bound proteins were eluted from the washed beads by boiling at 95°C in Laemmli buffer and subjected to SDS–PAGE and IB.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C). .. The immunoprecipitates were then resolved by 10% SDS-PAGE, and the T7-Impα3 that was pulled down together with GFP-IN was analyzed by Western blotting using mouse anti-T7 antibodies.

Plasmid Preparation:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: To detect the interaction between Impα3 and HIV IN, Vpr, MA, or different IN deletion mutants in mammalian cells, 293T cells were cotransfected with T7-Impα3 and the AcGFP-INwt/mut, MA-YFP, or Vpr-YFP expression plasmid. .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Co-Immunoprecipitation Assay:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Paragraph title: In vitro binding assay and cell-based coimmunoprecipitation (co-IP) experiments. ... Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Agarose Gel Electrophoresis:

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. Separation. (i) TAE agarose gel.

In Vitro:

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Paragraph title: In vitro binding assay and cell-based coimmunoprecipitation (co-IP) experiments. ... Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C).

Phosphorylation Assay:

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: Paragraph title: Dab1 Phosphorylation Assay ... After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 .

Immunoprecipitation:

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF. .. Protein concentrations were determined by BCA protein assay (Pierce) and were equalized among samples.

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿
Article Snippet: Paragraph title: Immunoprecipitation analysis. ... 293 cells were transfected with the indicated expression plasmids, and cell lysates were harvested in NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, 0.75% IPEGAL) containing protease inhibitor cocktail (Roche) 48 h posttransfection.

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: Paragraph title: Immunoprecipitation-Western assay ... The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C). .. After centrifugation, the clarified supernatant was subjected to immunoprecipitation using a rabbit anti-GFP antibody.

Lysis:

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: .. To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase.

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoprecipitation 107 cells/mL were pelleted by centrifugation, washed and resuspended in PBS and lysed on ice in equal volume of 2x NP-40 lysis buffer (40 mM Tris-HCL pH8, 274mM NaCl, 2% NP-40, 4mM EDTA) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 2mM DTT, 2mM Va3VO4 and 2mM PMSF. .. Protein concentrations were determined by BCA protein assay (Pierce) and were equalized among samples.

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: .. HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris. ..

Article Title: Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins
Article Snippet: .. The cells were pelleted by centrifugation (693 × g , 5 min, 4°C) and resuspended in 1 ml of NP-40 lysis buffer (1% 4-nonylphenolpolyethyleneglycol, 0.5% deoxycholate, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing protease inhibitors (protease inhibitor cocktail Complete; Roche). .. Following incubation on ice for 15 min, insoluble material was removed by centrifugation (16,000 × g , 30 min, 4°C).

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy
Article Snippet: .. After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na3 VO4 . .. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Article Title: The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner ▿
Article Snippet: .. 293 cells were transfected with the indicated expression plasmids, and cell lysates were harvested in NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, 0.75% IPEGAL) containing protease inhibitor cocktail (Roche) 48 h posttransfection. ..

Article Title: Productive Infection and bICP0 Early Promoter Activity of Bovine Herpesvirus 1 Are Stimulated by E2F1 ▿
Article Snippet: .. Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). .. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS.

Article Title: 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion
Article Snippet: .. Parasites were spun down and washed three times in PBS, and resuspended in 1% NP-40 lysis buffer containing Complete (Roche) protease inhibitors. ..

Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
Article Snippet: .. Co-Immunoprecipitation IFN-γ-induced RAW264.7 cells were lysed for 30 min at 4°C in 0.5% NP-40 lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and 0.5 mM GTP) and nuclear fraction was removed by centrifugation. .. Supernatants were pre-cleared for 3 h at 4°C with Protein G beads and subsequently incubated for 2 h at 4°C with a rat monoclonal anti-HA antibody (Roche Applied Science) and Protein G beads.

Article Title: VIRION INCORPORATION OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN VP22 IS FACILITATED BY TRANS GOLGI NETWORK LOCALIZATION AND IS INDEPENDENT OF INTERACTION WITH GLYCOPROTEIN E
Article Snippet: .. The 9 ml samples were lysed in NP-40 lysis buffer containing Complete Mini protease inhibitors as described above and precleared overnight at 4 °C with protein G-agarose beads (Roche) that had been washed twice in lysis buffer. .. The lysates were then incubated with a goat polyclonal antibody raised against GFP (Rockland) for 1 h at 4 °C, and immune complexes were collected with protein G-agarose beads that had been washed twice with lysis buffer.

Article Title: Importin ?3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication ▿
Article Snippet: .. Then 90% of the cells were lysed in NP-40 lysis buffer (199 medium containing 0.25% NP-40 Alternative and a protease inhibitor cocktail [Roche]) on ice for 30 min, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C). .. After centrifugation, the clarified supernatant was subjected to immunoprecipitation using a rabbit anti-GFP antibody.

Gradient Centrifugation:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: Paragraph title: Isolation of HBV NCs from cell lysates and virions by sucrose gradient centrifugation. ... HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris.

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  • 94
    Roche nondenaturing np 40 lysis buffer
    Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with <t>nondenaturing</t> <t>NP-40</t> lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.
    Nondenaturing Np 40 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nondenaturing np 40 lysis buffer/product/Roche
    Average 94 stars, based on 1 article reviews
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    99
    Roche np 40 lysis buffer
    CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with <t>NP-40</t> (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.
    Np 40 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np 40 lysis buffer/product/Roche
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    93
    Roche immunoprecipitation ip buffer
    Mode of inhibition of TNFR1 signaling by zafirlukast and triclabendazole (A) Effect of zafirlukast and triclabendazole (200 μM) on TNFR1-LTα interaction was determined by <t>co-immunoprecipitation</t> with anti-FLAG–conjugated agarose beads. (B) Oligomeric states of PLAD were determined by analytical size-exclusion chromatography. Dimer peak is indicated. Open triangle indicates non-specific protein. (C) Effect of zafirlukast and triclabendazole on PLAD-PLAD interactions was determined by Native-PAGE. Open circle indicates nonspecific band. (D) Docking scores for zafirlukast and triclabendazole on TNFR1 chain B (PDB: 1NCF) interfacial residues. Boltzmann-weighted scores were calculated by multiplying the probability of contacting a given residue by the mean Boltzmann-weighted predicted free energy for each pose contacting that residue.
    Immunoprecipitation Ip Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with nondenaturing NP-40 lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

    Journal: Infection and Immunity

    Article Title: Characterization of Pyrin Dephosphorylation and Inflammasome Activation in Macrophages as Triggered by the Yersinia Effectors YopE and YopT

    doi: 10.1128/IAI.00822-18

    Figure Lengend Snippet: Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with nondenaturing NP-40 lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

    Article Snippet: To obtain cell lysates, BMDMs were lysed using nondenaturing NP-40 lysis buffer (1% NP-40, 150 mM NaCl, and 50 mM Tris-HCl, pH 8.0) or denaturing RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid [DOC], and 0.1% SDS) with protease inhibitor cocktail (Roche).

    Techniques: Western Blot, De-Phosphorylation Assay, Infection, Plasmid Preparation, Lysis, Centrifugation

    CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

    Article Snippet: To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids.

    Techniques: Gradient Centrifugation, Southern Blot, Cell Culture, Incubation, Concentration Assay

    Isolation of HBV NCs from MNase-treated lysates by linear sucrose gradient centrifugation and detection of NC-associated viral DNA, RNA, and capsid protein. Induced HepAD38 cells were lysed by using NP-40, and the cytoplasmic lysate was treated with MNase.

    Journal: Journal of Virology

    Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

    doi: 10.1128/JVI.01912-13

    Figure Lengend Snippet: Isolation of HBV NCs from MNase-treated lysates by linear sucrose gradient centrifugation and detection of NC-associated viral DNA, RNA, and capsid protein. Induced HepAD38 cells were lysed by using NP-40, and the cytoplasmic lysate was treated with MNase.

    Article Snippet: HepAD38 cells induced for 20 days without Tet were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40) containing a protease inhibitor cocktail (Roche) and briefly centrifuged at 14,000 rpm to remove the nuclei and cell debris.

    Techniques: Isolation, Gradient Centrifugation

    Mode of inhibition of TNFR1 signaling by zafirlukast and triclabendazole (A) Effect of zafirlukast and triclabendazole (200 μM) on TNFR1-LTα interaction was determined by co-immunoprecipitation with anti-FLAG–conjugated agarose beads. (B) Oligomeric states of PLAD were determined by analytical size-exclusion chromatography. Dimer peak is indicated. Open triangle indicates non-specific protein. (C) Effect of zafirlukast and triclabendazole on PLAD-PLAD interactions was determined by Native-PAGE. Open circle indicates nonspecific band. (D) Docking scores for zafirlukast and triclabendazole on TNFR1 chain B (PDB: 1NCF) interfacial residues. Boltzmann-weighted scores were calculated by multiplying the probability of contacting a given residue by the mean Boltzmann-weighted predicted free energy for each pose contacting that residue.

    Journal: SLAS discovery : advancing life sciences R & D

    Article Title: An innovative high-throughput screening approach for discovery of small molecules that inhibit TNF Receptors

    doi: 10.1177/2472555217706478

    Figure Lengend Snippet: Mode of inhibition of TNFR1 signaling by zafirlukast and triclabendazole (A) Effect of zafirlukast and triclabendazole (200 μM) on TNFR1-LTα interaction was determined by co-immunoprecipitation with anti-FLAG–conjugated agarose beads. (B) Oligomeric states of PLAD were determined by analytical size-exclusion chromatography. Dimer peak is indicated. Open triangle indicates non-specific protein. (C) Effect of zafirlukast and triclabendazole on PLAD-PLAD interactions was determined by Native-PAGE. Open circle indicates nonspecific band. (D) Docking scores for zafirlukast and triclabendazole on TNFR1 chain B (PDB: 1NCF) interfacial residues. Boltzmann-weighted scores were calculated by multiplying the probability of contacting a given residue by the mean Boltzmann-weighted predicted free energy for each pose contacting that residue.

    Article Snippet: HEK293 and transiently transfected cells were washed three times with ice-cold PBS before lysis with immunoprecipitation (IP) buffer (20 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.5 and 0.5% NP-40) containing complete protease inhibitors cocktail (Roche).

    Techniques: Inhibition, Immunoprecipitation, Size-exclusion Chromatography, Clear Native PAGE