Structured Review

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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 <t>NP-40</t> 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.
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Images

1) 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

2) Product Images from "Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †"

Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.00573-10

Coexpression of LF2 results in redistribution of Rta out of the nucleus to the cytoskeleton. (A) Crude cytoplasmic nuclear fractionation and subsequent NP-40 extraction of the nuclear fraction. 293T cells were transfected with Rta, Flag-LF2, or both,
Figure Legend Snippet: Coexpression of LF2 results in redistribution of Rta out of the nucleus to the cytoskeleton. (A) Crude cytoplasmic nuclear fractionation and subsequent NP-40 extraction of the nuclear fraction. 293T cells were transfected with Rta, Flag-LF2, or both,

Techniques Used: Fractionation, Transfection

3) Product Images from "Loss of Tyrosine Phosphatase Dependent Inhibition Promotes Activation of Tyrosine Kinase c-Src in Detached Pancreatic Cells"

Article Title: Loss of Tyrosine Phosphatase Dependent Inhibition Promotes Activation of Tyrosine Kinase c-Src in Detached Pancreatic Cells

Journal: Molecular carcinogenesis

doi: 10.1002/mc.20684

(A) SHP-2 inhibitor NSC-87877 abrogates detachment-mediated Src activation. Adherent L3.6pl cells were treated with 0 or 50 μM NSC-87877 for three hours and either lysed or detached via trypsinization. Detached cells were incubated for 20 min in the presence or absence of 50 μM NSC-87877. Src activity was evaluated via anti-phospho-Src Y418 western blot analysis. Blots were stripped and re-probed for total Src. The graph represents the ratio of phospho-Src Y418 to total Src, relative to untreated, attached cells (N=3). (B) Co-association of Src and SHP-2 in detached pancreatic cancer cells. Attached and detached (20 min) L3.6pl or PANC-1 cells were lysed in NP-40. Src was immunoprecipitated as described in Materials and Methods, and co-immunoprecipitating SHP-2 was detected by western blot analysis. Blots were stripped and re-probed for total Src. Whole cell lysates were probed for phospho-SHP-2 Y542 , stripped, and re-probed for total SHP-2. The graphs represent the ratio of immunoprecipitated SHP-2 to Src, relative to attached cells (N=3).
Figure Legend Snippet: (A) SHP-2 inhibitor NSC-87877 abrogates detachment-mediated Src activation. Adherent L3.6pl cells were treated with 0 or 50 μM NSC-87877 for three hours and either lysed or detached via trypsinization. Detached cells were incubated for 20 min in the presence or absence of 50 μM NSC-87877. Src activity was evaluated via anti-phospho-Src Y418 western blot analysis. Blots were stripped and re-probed for total Src. The graph represents the ratio of phospho-Src Y418 to total Src, relative to untreated, attached cells (N=3). (B) Co-association of Src and SHP-2 in detached pancreatic cancer cells. Attached and detached (20 min) L3.6pl or PANC-1 cells were lysed in NP-40. Src was immunoprecipitated as described in Materials and Methods, and co-immunoprecipitating SHP-2 was detected by western blot analysis. Blots were stripped and re-probed for total Src. Whole cell lysates were probed for phospho-SHP-2 Y542 , stripped, and re-probed for total SHP-2. The graphs represent the ratio of immunoprecipitated SHP-2 to Src, relative to attached cells (N=3).

Techniques Used: Activation Assay, Incubation, Activity Assay, Western Blot, Immunoprecipitation

Effects of Src family kinase inhibition on detachment-dependent activation of Erk-1/2, Akt, and JNK. Attached and detached (indicated times) L3.6pl pancreatic cancer cells were treated with 0 or 1 μM AP23846 as described in Materials and Methods. Cells were lysed in NP-40 buffer. Cell lysates were resolved by 8% SDS-PAGE and western blot analysis was performed for phospho-Erk-1/2 T202/Y204 (A), phospho-Akt S473 (B), or phospho-JNK T183/Y186 (C). Blots were stripped and re-probed for total Erk-1/2 (A), total Akt (B), or total JNK (C) respectively. The graphs represent the ratio of phospho-Erk-1/2 T202/Y204 (A), phospho- Akt S473 (B), and phospho- JNK T183/Y186 (C) to total Erk-1/2, Akt, and JNK, respectively (N=3).
Figure Legend Snippet: Effects of Src family kinase inhibition on detachment-dependent activation of Erk-1/2, Akt, and JNK. Attached and detached (indicated times) L3.6pl pancreatic cancer cells were treated with 0 or 1 μM AP23846 as described in Materials and Methods. Cells were lysed in NP-40 buffer. Cell lysates were resolved by 8% SDS-PAGE and western blot analysis was performed for phospho-Erk-1/2 T202/Y204 (A), phospho-Akt S473 (B), or phospho-JNK T183/Y186 (C). Blots were stripped and re-probed for total Erk-1/2 (A), total Akt (B), or total JNK (C) respectively. The graphs represent the ratio of phospho-Erk-1/2 T202/Y204 (A), phospho- Akt S473 (B), and phospho- JNK T183/Y186 (C) to total Erk-1/2, Akt, and JNK, respectively (N=3).

Techniques Used: Inhibition, Activation Assay, SDS Page, Western Blot

Activation of Erk-1/2, Akt, and JNK in detached L3.6pl pancreatic cancer cells. Adherent, detached (indicated times), and reattached (indicated times) L3.6pl cells were lysed in NP-40 buffer. Cell lysates were resolved by 8% SDS-PAGE and western blot analysis was performed for phospho-Erk-1/2 T202/Y204 (A), phospho-Akt S473 (B), or phospho-JNK T183/Y186 (C). Blots were stripped and re-probed for total Erk-1/2 (A), total Akt (B), or total JNK (C) respectively. The graphs represent the ratio of phospho-Erk-1/2 T202/Y204 (A), phospho- Akt S473 (B), and phospho- JNK T183/Y186 (C) to total Erk-1/2, Akt, and JNK, respectively (N=3).
Figure Legend Snippet: Activation of Erk-1/2, Akt, and JNK in detached L3.6pl pancreatic cancer cells. Adherent, detached (indicated times), and reattached (indicated times) L3.6pl cells were lysed in NP-40 buffer. Cell lysates were resolved by 8% SDS-PAGE and western blot analysis was performed for phospho-Erk-1/2 T202/Y204 (A), phospho-Akt S473 (B), or phospho-JNK T183/Y186 (C). Blots were stripped and re-probed for total Erk-1/2 (A), total Akt (B), or total JNK (C) respectively. The graphs represent the ratio of phospho-Erk-1/2 T202/Y204 (A), phospho- Akt S473 (B), and phospho- JNK T183/Y186 (C) to total Erk-1/2, Akt, and JNK, respectively (N=3).

Techniques Used: Activation Assay, SDS Page, Western Blot

(A) Src Y418 phosphorylation in attached and detached pancreatic cancer cells is dependent on Src activity. PANC-1 cells were treated for one hour with 0 or 1 μM AP23846. After one hour, cells were lysed in NP-40 buffer or detached via trypsinization and maintained in suspension for 0 or 20 min at 37 °C in the presence of 0 or 1 μM AP23846 prior to lysis. Src was immunoprecipitated from whole cell lysates, and western blot analysis was performed for anti-phospho-Src Y418 . Blots were stripped and re-probed for total Src. (B) Activation of Src in detached L3.6pl cells, relative to cells adherent to extracellular matrix proteins. L3.6pl cells were released via trypsinization, rotated at 37 °C for 20 min, and re-plated on standard 100 mm tissue culture plastic (TCP) or dishes pre-coated with poly-D-lysine, collagen I, fibronectin, or laminin. Adherent, detached, and reattached cells were lysed in NP-40-buffer, and Src activity was evaluated as described in (A). All blots are representative of at least three experiments.
Figure Legend Snippet: (A) Src Y418 phosphorylation in attached and detached pancreatic cancer cells is dependent on Src activity. PANC-1 cells were treated for one hour with 0 or 1 μM AP23846. After one hour, cells were lysed in NP-40 buffer or detached via trypsinization and maintained in suspension for 0 or 20 min at 37 °C in the presence of 0 or 1 μM AP23846 prior to lysis. Src was immunoprecipitated from whole cell lysates, and western blot analysis was performed for anti-phospho-Src Y418 . Blots were stripped and re-probed for total Src. (B) Activation of Src in detached L3.6pl cells, relative to cells adherent to extracellular matrix proteins. L3.6pl cells were released via trypsinization, rotated at 37 °C for 20 min, and re-plated on standard 100 mm tissue culture plastic (TCP) or dishes pre-coated with poly-D-lysine, collagen I, fibronectin, or laminin. Adherent, detached, and reattached cells were lysed in NP-40-buffer, and Src activity was evaluated as described in (A). All blots are representative of at least three experiments.

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

(A) Phosphorylation of Src on Tyr 529 in detached L3.6pl pancreatic cancer cells. Adherent, detached (indicated times), and reattached (indicated times) L3.6pl cells were lysed in NP-40 buffer. Total Src was immunoprecipitated, and anti-phospho-Src Y529 western blot analysis was performed. Blots were stripped and re-probed for total Src. The graph represents the ratio of phospho-Src Y529 to total Src, relative to attached cells (N=3). (B) Autophosphorylation of Src in the presence of the phosphatase inhibitor sodium vanadate. L3.6pl cells were treated with 0 or 50 μM sodium vanadate for one hour. Cells were lysed, and total Src was immunoprecipitated. Anti-phospho-Src Y418 western blot analysis was performed. Blots were stripped and re-probed for phospho-Src Y529 and total Src. The graphs represent the ratio of phospho-Src Y418 or phospho-Src Y529 , as indicated, to total Src, relative to untreated cells (N=3).
Figure Legend Snippet: (A) Phosphorylation of Src on Tyr 529 in detached L3.6pl pancreatic cancer cells. Adherent, detached (indicated times), and reattached (indicated times) L3.6pl cells were lysed in NP-40 buffer. Total Src was immunoprecipitated, and anti-phospho-Src Y529 western blot analysis was performed. Blots were stripped and re-probed for total Src. The graph represents the ratio of phospho-Src Y529 to total Src, relative to attached cells (N=3). (B) Autophosphorylation of Src in the presence of the phosphatase inhibitor sodium vanadate. L3.6pl cells were treated with 0 or 50 μM sodium vanadate for one hour. Cells were lysed, and total Src was immunoprecipitated. Anti-phospho-Src Y418 western blot analysis was performed. Blots were stripped and re-probed for phospho-Src Y529 and total Src. The graphs represent the ratio of phospho-Src Y418 or phospho-Src Y529 , as indicated, to total Src, relative to untreated cells (N=3).

Techniques Used: Immunoprecipitation, Western Blot

(A) Activation of Src in detached L3.6pl pancreatic cancer cells. Adherent, detached (indicated times), and reattached (indicated times) L3.6pl cells were lysed in NP-40 buffer. Total Src was immunoprecipitated, and Src activity was visualized via anti-phospho-Src Y418 western blot analysis. Blots were stripped and re-probed for total Src. The graph represents the ratio of phospho-Src Y418 staining to total Src, relative to attached cells (N=3). (B) Detachment-specific activation of Src in a panel of pancreatic cancer cell lines. Adherent, suspended (20 min), and reattached (60 min) L3.6pl, Hs766T, PANC-1, and BxPC3 cells were lysed in NP-40 buffer. Src was immunoprecipitated and anti-phospho-Src Y418 western blot analysis was performed as described in Materials and Methods. Blots were stripped and re-probed for total Src. (C) Activation of Yes in detached pancreatic cancer cells. Adherent, suspended (20 min), or reattached (60 min) L3.6pl cells were lysed in NP-40. Total Yes was immunoprecipitated, and Yes activity was visualized via anti-phospho-Src Y418 western blot analysis. Blots were stripped and re-probed for total Yes. The graph represents the ratio of phospho-Yes to total Yes, relative to attached cells (N=3).
Figure Legend Snippet: (A) Activation of Src in detached L3.6pl pancreatic cancer cells. Adherent, detached (indicated times), and reattached (indicated times) L3.6pl cells were lysed in NP-40 buffer. Total Src was immunoprecipitated, and Src activity was visualized via anti-phospho-Src Y418 western blot analysis. Blots were stripped and re-probed for total Src. The graph represents the ratio of phospho-Src Y418 staining to total Src, relative to attached cells (N=3). (B) Detachment-specific activation of Src in a panel of pancreatic cancer cell lines. Adherent, suspended (20 min), and reattached (60 min) L3.6pl, Hs766T, PANC-1, and BxPC3 cells were lysed in NP-40 buffer. Src was immunoprecipitated and anti-phospho-Src Y418 western blot analysis was performed as described in Materials and Methods. Blots were stripped and re-probed for total Src. (C) Activation of Yes in detached pancreatic cancer cells. Adherent, suspended (20 min), or reattached (60 min) L3.6pl cells were lysed in NP-40. Total Yes was immunoprecipitated, and Yes activity was visualized via anti-phospho-Src Y418 western blot analysis. Blots were stripped and re-probed for total Yes. The graph represents the ratio of phospho-Yes to total Yes, relative to attached cells (N=3).

Techniques Used: Activation Assay, Immunoprecipitation, Activity Assay, Western Blot, Staining

4) Product Images from "Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion"

Article Title: Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20051027

Crk stimulates CagA-dependent breakdown of cell–cell adhesion. (A) Lysates from MKN74 cells transfected for 72 h with Luc-siRNA or Crk-siRNA at 200 nM were subjected to immunoblotting with the indicated antibodies. (B) MKN74 cells treated for 72 h with Luc-RNAi (a–d) or Crk/Crk-L-RNAi (e–h) were mock infected (a and e) or infected with the H. pylori Δ vacA /Δ cagA (b and f) or Δ vacA strain (c, d, g, and h) at an MOI of 20. The cells were fixed 48 h after infection and stained with anti–β-catenin antibody. White arrowheads (d and h) point to the nuclei of the cells. Note that Δ vacA H. pylori strains were used to reduce damage to the infected cells (references 1 , 60 ). Bar, 20 μm. (C) NP-40–soluble (S) and –insoluble fractions (IS) were prepared from H. pylori –infected cells in B, and the fractions were subjected to immunoblotting with the indicated antibodies. (D) The blots of NP-40–soluble fractions in C were quantified by densitometry. The data are presented as fold increase in amounts of Δ vacA compared with that of Δ vacA /Δ cagA under the same RNAi conditions. The data shown represent means and SDs of triplicate experiments.
Figure Legend Snippet: Crk stimulates CagA-dependent breakdown of cell–cell adhesion. (A) Lysates from MKN74 cells transfected for 72 h with Luc-siRNA or Crk-siRNA at 200 nM were subjected to immunoblotting with the indicated antibodies. (B) MKN74 cells treated for 72 h with Luc-RNAi (a–d) or Crk/Crk-L-RNAi (e–h) were mock infected (a and e) or infected with the H. pylori Δ vacA /Δ cagA (b and f) or Δ vacA strain (c, d, g, and h) at an MOI of 20. The cells were fixed 48 h after infection and stained with anti–β-catenin antibody. White arrowheads (d and h) point to the nuclei of the cells. Note that Δ vacA H. pylori strains were used to reduce damage to the infected cells (references 1 , 60 ). Bar, 20 μm. (C) NP-40–soluble (S) and –insoluble fractions (IS) were prepared from H. pylori –infected cells in B, and the fractions were subjected to immunoblotting with the indicated antibodies. (D) The blots of NP-40–soluble fractions in C were quantified by densitometry. The data are presented as fold increase in amounts of Δ vacA compared with that of Δ vacA /Δ cagA under the same RNAi conditions. The data shown represent means and SDs of triplicate experiments.

Techniques Used: Transfection, Infection, Staining

5) Product Images from "TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer"

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-0829

TBK1 supports mTORC1 pathway activation both upstream and downstream of mTOR (A) Inhibition of TBK1 or AKT similarly blunts mTORC1 activation alone or together in PDPK1 -null (−/−) HCT116 cells. Cells were starved in EBSS for 3 hrs in the presence of DMSO or 0.5µM MK2206 (allosteric AKT inhibitor). Following starvation, but prior to EAA addition at the indicated times, cells were treated with DMSO or 0.5µM BX795 in EBSS for 30 min. mTORC1 phosphorylation of S6K (pT389) and A KT phosphorylation of PRAS40 (pT246) were assessed via immunoblot. Densitometry ratios for S6K (pT389)/S6K (Total) were calculated via ImageJ and are listed relative to the 20 min EAA time point in the DMSO-treated condition. (B) TBK1 loss of function corresponds to increased PRAS40-mTORC1 association. Endogenous Raptor was immunoprecipitated from Tbk1 WT or (Δ/Δ) MEFs. Elevated affinity of PRAS40 for Raptor in (Δ/Δ) MEFs was maintained under (Left) 0.3% CHAPS or (Right) 0.5% NP-40 cell lysis conditions. Control = IP with an anti-FLAG-tag antibody. (C) TBK1 inhibitors partially blunt AKT/mTORC1 signaling in Tsc2 WT or homozygous knock-out (−/−) Trp53 −/− immortalized MEFs. DMSO or 2µM Compound II, BX795 or GSK2292978A (GSK) was added after starvation, 30 min before addition of EAA for the indicated times. (D) TBK1 is required for an amino acid-induced S6K C-terminal autoinhibitory domain phosphorylation at pT421/S424. Loss of TBK1 also attenuated TBK1 autophosphorylation (pS172) and the activating phosphorylation of S6K by mTORC1 (pT389), but not PDK1 (pT229) upon EAA stimulation in Tbk1 −/− MEFs (E) S6K is phosphorylated in its autoinhibitory domain in a TBK1 kinase-dependent manner in TBK1 complexes. Myc-vector-only, myc-FLAG-TBK1-WT, myc-FLAG-TBK1-KD, or HA-TBK1 were overexpressed in HEK293FT cells, followed by anti-FLAG IP. WCL = whole cell lysate. (F) TBK1 can phosphorylate the autoinhibitory domain of S6K in vitro . Recombinant TBK1 and S6K (pretreated with lambda phosphatase) were incubated either alone or together in the presence or absence of 200µM ATP. ATP-dependent phosphorylation of TBK1 and S6K were assessed by immunoblot as indicated. (G) TBK1 physically and functionally interacts with multiple AKT/mTORC1 regulatory elements. In this cartoon, green arrows point toward nodes whose activity is directly or indirectly promoted by TBK1, whereas grey lines indicate points of physical association with TBK1.
Figure Legend Snippet: TBK1 supports mTORC1 pathway activation both upstream and downstream of mTOR (A) Inhibition of TBK1 or AKT similarly blunts mTORC1 activation alone or together in PDPK1 -null (−/−) HCT116 cells. Cells were starved in EBSS for 3 hrs in the presence of DMSO or 0.5µM MK2206 (allosteric AKT inhibitor). Following starvation, but prior to EAA addition at the indicated times, cells were treated with DMSO or 0.5µM BX795 in EBSS for 30 min. mTORC1 phosphorylation of S6K (pT389) and A KT phosphorylation of PRAS40 (pT246) were assessed via immunoblot. Densitometry ratios for S6K (pT389)/S6K (Total) were calculated via ImageJ and are listed relative to the 20 min EAA time point in the DMSO-treated condition. (B) TBK1 loss of function corresponds to increased PRAS40-mTORC1 association. Endogenous Raptor was immunoprecipitated from Tbk1 WT or (Δ/Δ) MEFs. Elevated affinity of PRAS40 for Raptor in (Δ/Δ) MEFs was maintained under (Left) 0.3% CHAPS or (Right) 0.5% NP-40 cell lysis conditions. Control = IP with an anti-FLAG-tag antibody. (C) TBK1 inhibitors partially blunt AKT/mTORC1 signaling in Tsc2 WT or homozygous knock-out (−/−) Trp53 −/− immortalized MEFs. DMSO or 2µM Compound II, BX795 or GSK2292978A (GSK) was added after starvation, 30 min before addition of EAA for the indicated times. (D) TBK1 is required for an amino acid-induced S6K C-terminal autoinhibitory domain phosphorylation at pT421/S424. Loss of TBK1 also attenuated TBK1 autophosphorylation (pS172) and the activating phosphorylation of S6K by mTORC1 (pT389), but not PDK1 (pT229) upon EAA stimulation in Tbk1 −/− MEFs (E) S6K is phosphorylated in its autoinhibitory domain in a TBK1 kinase-dependent manner in TBK1 complexes. Myc-vector-only, myc-FLAG-TBK1-WT, myc-FLAG-TBK1-KD, or HA-TBK1 were overexpressed in HEK293FT cells, followed by anti-FLAG IP. WCL = whole cell lysate. (F) TBK1 can phosphorylate the autoinhibitory domain of S6K in vitro . Recombinant TBK1 and S6K (pretreated with lambda phosphatase) were incubated either alone or together in the presence or absence of 200µM ATP. ATP-dependent phosphorylation of TBK1 and S6K were assessed by immunoblot as indicated. (G) TBK1 physically and functionally interacts with multiple AKT/mTORC1 regulatory elements. In this cartoon, green arrows point toward nodes whose activity is directly or indirectly promoted by TBK1, whereas grey lines indicate points of physical association with TBK1.

Techniques Used: Activation Assay, Inhibition, Immunoprecipitation, Lysis, FLAG-tag, Knock-Out, Plasmid Preparation, In Vitro, Recombinant, Incubation, Activity Assay

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Nucleic Acid Electrophoresis:

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Transfection:

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Incubation:

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In Vitro:

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Immunoprecipitation:

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Article Snippet: Paragraph title: Immunoprecipitation and pull-down experiments ... Cell pellets were resuspended in 10mls NP-40 Buffer (10 mM sodium phosphate, pH8, 150 mM NaCl, 1% Nonidet P-40, and Roche EDTA-free Complete protease inhibitor cocktail) and lysed using a Constant Systems Cell Disruptor (4 passes at 40k psi), and spun at 50,000g for 30 minutes to generate lysate.

Article Title: Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer
Article Snippet: Paragraph title: GST pulldown, immunoblot, and immunoprecipitation analysis ... For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche).

Protease Inhibitor:

Article Title: Zeb2 Recruits HDAC-NuRD to Inhibit Notch and Controls Schwann Cell Differentiation and Remyelination
Article Snippet: .. Cells were lysed in NP-40 buffer (170 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.4, 50 mM NaF, and 0.5% NP-40) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Roche Diagnostics Inc.). .. A total of 300 μg of cell lysate proteins were incubated with 2 μg IgG or appropriate antibodies and immunoprecipitated using Protein A/G beads (Santa Cruz Biotechnology).

Article Title: Regulation of p53 stability and function by the deubiquitinating enzyme USP42
Article Snippet: .. Cell extracts were prepared in either NP-40 buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40) or RIPA buffer (50 mM Tris–HCl, pH 8.0, 120 mM NaCl, 1% NP-40, 1% SDS 0.5% DOC, protease inhibitor cocktail (Roche)). .. Proteins were resolved by SDS–PAGE and transferred onto nitrocellulose membrane (Millipore).

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10
Article Snippet: .. THP-1 WT cells were lysed in Buffer L (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 5.5, and Roche cOmplete protease inhibitor cocktail) or 1% IGEPAL CA-630 buffer (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 7.4, and Roche cOmplete protease inhibitor cocktail). .. Lysate was clarified by centrifugation at 12,500 × g for 5 min. Biotin-labeled poly(I:C) (used at 5 ng/ml, average size: 1.5–8.0 kb) was added to the lysate with or without addition of unlabeled competitive poly(I:C) (50 ng/ml, average size: 1.5–8.0 kb).

Article Title: Kaposi's Sarcoma-Associated Herpesvirus K7 Protein Targets a Ubiquitin-Like/Ubiquitin-Associated Domain-Containing Protein To Promote Protein Degradation
Article Snippet: .. Cells were harvested and then lysed with NP-40 buffer (0.15 M NaCl, 1% Nonidet P-40, 50 mM Tris, pH 7.5) containing 0.1 mM Na2 VO3 , 1 mM NaF, and protease inhibitor cocktail (Roche, Mannheim, Germany). .. In order to demonstrate PLIC dimerization, cells were lysed with phosphate-buffered saline (pH 7.4) (Sigma) supplemented with NP-40 to a final concentration of 0.2% and protease inhibitor cocktail.

Article Title: Sox11 Reduces Caspase-6 Cleavage and Activity
Article Snippet: .. Cells were harvested 24hours post-transfection and lysed on ice using an NP-40 buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.02% NaN3 ) supplemented with complete protease inhibitor (Roche). .. Protein concentration was determined by the BCA method (Pierce)

Article Title: Loss of Tyrosine Phosphatase Dependent Inhibition Promotes Activation of Tyrosine Kinase c-Src in Detached Pancreatic Cells
Article Snippet: .. Adherent cells were washed twice with ice-cold tris-buffered saline (TBS), followed by lysis for 10 min in NP-40 buffer (150 mM NaCl, 5 mM EDTA, 1% NP-40, pH 7.4), supplemented with one tablet complete mini-EDTA-free protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany), and sodium orthovanadate (1 mM, pH 7.4). .. Cells were scraped from plates, and cell lysates were clarified by centrifugation for 5 min at 13,000 × g and 4 °C.

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoblot Cells were pelleted by centrifugation, washed with ice-cold PBS and lysed on ice for 20 min in modified NP-40 buffer (10mM/1mM KPO4/EDTA pH 7.05, 5mM EGTA pH 7.2, 10mM MgCl2, 50mM glycerophosphate pH 7.2, 0.5% NP-40, 0.1% Brij-35) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 1mM DTT, 1mM Va3VO4 and 1mM PMSF. .. Protein concentrations of whole-cell lysates were determined by BCA protein assays (Pierce), and equivalent protein amounts (20-60 μg) were electrophoresed through 4-12% Bis-Tris acrylamide gels.

Article Title: Rv0004 is a new essential member of the mycobacterial DNA replication machinery
Article Snippet: .. Cell pellets were resuspended in 10mls NP-40 Buffer (10 mM sodium phosphate, pH8, 150 mM NaCl, 1% Nonidet P-40, and Roche EDTA-free Complete protease inhibitor cocktail) and lysed using a Constant Systems Cell Disruptor (4 passes at 40k psi), and spun at 50,000g for 30 minutes to generate lysate. .. When indicated, some lysates were treated with DNase I (NEB) according to manufacturers instructions.

Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
Article Snippet: .. The pellet fraction was lysed with isotonic NP-40 buffer (1% [vol/vol] Igepal CA-630, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 5 mM MgCl2 ) containing Complete protease inhibitor (Roche). .. Two percent of the NP-40-soluble lysate and of the NP-40-insoluble pellet were analyzed by Western blotting.

Article Title: Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer
Article Snippet: .. For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). .. After centrifugation, the supernatants were precleared with protein A/G beads at 4°C for 2 h. Pre-cleared lysates were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was incubated for 4 h at 4°C.

Cell Fractionation:

Article Title: Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion
Article Snippet: .. Cell fractionation 1% NP-40–soluble and –insoluble fractions were prepared by lysing cells in NP-40 buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1% NP-40, 1 mM Na3 VO4 , and complete protease inhibitors; Roche) by 10 passages through a 27-gauge syringe and allowing to then stand at 4°C for 30 min. ..

Centrifugation:

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10
Article Snippet: THP-1 WT cells were lysed in Buffer L (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 5.5, and Roche cOmplete protease inhibitor cocktail) or 1% IGEPAL CA-630 buffer (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 7.4, and Roche cOmplete protease inhibitor cocktail). .. Lysate was clarified by centrifugation at 12,500 × g for 5 min. Biotin-labeled poly(I:C) (used at 5 ng/ml, average size: 1.5–8.0 kb) was added to the lysate with or without addition of unlabeled competitive poly(I:C) (50 ng/ml, average size: 1.5–8.0 kb).

Article Title: Loss of Tyrosine Phosphatase Dependent Inhibition Promotes Activation of Tyrosine Kinase c-Src in Detached Pancreatic Cells
Article Snippet: Adherent cells were washed twice with ice-cold tris-buffered saline (TBS), followed by lysis for 10 min in NP-40 buffer (150 mM NaCl, 5 mM EDTA, 1% NP-40, pH 7.4), supplemented with one tablet complete mini-EDTA-free protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany), and sodium orthovanadate (1 mM, pH 7.4). .. Cells were scraped from plates, and cell lysates were clarified by centrifugation for 5 min at 13,000 × g and 4 °C.

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoblot Cells were pelleted by centrifugation, washed with ice-cold PBS and lysed on ice for 20 min in modified NP-40 buffer (10mM/1mM KPO4/EDTA pH 7.05, 5mM EGTA pH 7.2, 10mM MgCl2, 50mM glycerophosphate pH 7.2, 0.5% NP-40, 0.1% Brij-35) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 1mM DTT, 1mM Va3VO4 and 1mM PMSF. .. Protein concentrations of whole-cell lysates were determined by BCA protein assays (Pierce), and equivalent protein amounts (20-60 μg) were electrophoresed through 4-12% Bis-Tris acrylamide gels.

Article Title: Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer
Article Snippet: For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). .. After centrifugation, the supernatants were precleared with protein A/G beads at 4°C for 2 h. Pre-cleared lysates were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was incubated for 4 h at 4°C.

Cell Culture:

Article Title: Zeb2 Recruits HDAC-NuRD to Inhibit Notch and Controls Schwann Cell Differentiation and Remyelination
Article Snippet: For immunoprecipitation, HEK293T cells cultured in 10% FBS/DMEM were transfected with expression vectors using PolyJet (Signagen) for 48 hours. .. Cells were lysed in NP-40 buffer (170 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.4, 50 mM NaF, and 0.5% NP-40) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Roche Diagnostics Inc.).

Article Title: Sox11 Reduces Caspase-6 Cleavage and Activity
Article Snippet: Paragraph title: Cell Culture and Transfections ... Cells were harvested 24hours post-transfection and lysed on ice using an NP-40 buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.02% NaN3 ) supplemented with complete protease inhibitor (Roche).

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: The next day, cell culture media was aspirated and 2mL of 1X Earle's Balanced Salt Solution (EBSS) was added for 3 hrs. .. All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol).

Concentration Assay:

Article Title: Kaposi's Sarcoma-Associated Herpesvirus K7 Protein Targets a Ubiquitin-Like/Ubiquitin-Associated Domain-Containing Protein To Promote Protein Degradation
Article Snippet: Cells were harvested and then lysed with NP-40 buffer (0.15 M NaCl, 1% Nonidet P-40, 50 mM Tris, pH 7.5) containing 0.1 mM Na2 VO3 , 1 mM NaF, and protease inhibitor cocktail (Roche, Mannheim, Germany). .. In order to demonstrate PLIC dimerization, cells were lysed with phosphate-buffered saline (pH 7.4) (Sigma) supplemented with NP-40 to a final concentration of 0.2% and protease inhibitor cocktail.

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: MEM-Essential Amino Acids Solution (EAA) (Sigma Aldrich) was added to a final concentration per well of 1X for the times listed. .. All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol).

Generated:

Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
Article Snippet: The pellet fraction was lysed with isotonic NP-40 buffer (1% [vol/vol] Igepal CA-630, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 5 mM MgCl2 ) containing Complete protease inhibitor (Roche). .. Subcellular fractions were also generated using a commercially available sequential extraction procedure (Pierce, Rockford, IL) according to the manufacturer's instructions.

Fractionation:

Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
Article Snippet: Paragraph title: Subcellular fractionation. ... The pellet fraction was lysed with isotonic NP-40 buffer (1% [vol/vol] Igepal CA-630, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 5 mM MgCl2 ) containing Complete protease inhibitor (Roche).

Expressing:

Article Title: Zeb2 Recruits HDAC-NuRD to Inhibit Notch and Controls Schwann Cell Differentiation and Remyelination
Article Snippet: For immunoprecipitation, HEK293T cells cultured in 10% FBS/DMEM were transfected with expression vectors using PolyJet (Signagen) for 48 hours. .. Cells were lysed in NP-40 buffer (170 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.4, 50 mM NaF, and 0.5% NP-40) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Roche Diagnostics Inc.).

Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
Article Snippet: A 0.5-μg aliquot of LF2 and 0.5 μg of Rta expression plasmids were transfected with Effectene into 293T cells. .. The pellet fraction was lysed with isotonic NP-40 buffer (1% [vol/vol] Igepal CA-630, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 5 mM MgCl2 ) containing Complete protease inhibitor (Roche).

Protein Concentration:

Article Title: Sox11 Reduces Caspase-6 Cleavage and Activity
Article Snippet: Cells were harvested 24hours post-transfection and lysed on ice using an NP-40 buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.02% NaN3 ) supplemented with complete protease inhibitor (Roche). .. Protein concentration was determined by the BCA method (Pierce)

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol). .. Following lysis on ice at 4C, SDS lysates were heated at ≥ 95C for ≥ 5 min and then protein concentration was measured via Pierce BCA assay.

Modification:

Article Title: Sox11 Reduces Caspase-6 Cleavage and Activity
Article Snippet: Cell Culture and Transfections HEK 293FT cells were maintained in Dulbecco’s modified media (Gibco) containing 10% FCS (Quality Biological), 0.5mg/ml G418 sulfate (Cellgro) and 1x penicillin/streptomycin (Invitrogen), in a humidified 5% CO2 incubator at 37°C. .. Cells were harvested 24hours post-transfection and lysed on ice using an NP-40 buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.02% NaN3 ) supplemented with complete protease inhibitor (Roche).

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: .. Immunoblot Cells were pelleted by centrifugation, washed with ice-cold PBS and lysed on ice for 20 min in modified NP-40 buffer (10mM/1mM KPO4/EDTA pH 7.05, 5mM EGTA pH 7.2, 10mM MgCl2, 50mM glycerophosphate pH 7.2, 0.5% NP-40, 0.1% Brij-35) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 1mM DTT, 1mM Va3VO4 and 1mM PMSF. .. Protein concentrations of whole-cell lysates were determined by BCA protein assays (Pierce), and equivalent protein amounts (20-60 μg) were electrophoresed through 4-12% Bis-Tris acrylamide gels.

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol). .. Protein concentration was equilibrated across experimental samples and Western Blot samples were prepared using 2X Sample Buffer (+β-me) and heated to ≥ 95C for ≥ 5 min. NP40 lysates were lysed for 15 min at 4C and then centrifuged at 20,000g for 20 min at 4C, and the protein concentration of the supernatant was quantified using Advanced Protein Assay Reagent (Cytoskeleton, modified Method 4).

Western Blot:

Article Title: Zeb2 Recruits HDAC-NuRD to Inhibit Notch and Controls Schwann Cell Differentiation and Remyelination
Article Snippet: Paragraph title: Western Blotting and Co-Immunoprecipitation ... Cells were lysed in NP-40 buffer (170 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.4, 50 mM NaF, and 0.5% NP-40) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Roche Diagnostics Inc.).

Article Title: Regulation of p53 stability and function by the deubiquitinating enzyme USP42
Article Snippet: Cell extracts were prepared in either NP-40 buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40) or RIPA buffer (50 mM Tris–HCl, pH 8.0, 120 mM NaCl, 1% NP-40, 1% SDS 0.5% DOC, protease inhibitor cocktail (Roche)). .. Immunoblots were quantitated using ImageJ software or the Licor Odyssey system.

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10
Article Snippet: THP-1 WT cells were lysed in Buffer L (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 5.5, and Roche cOmplete protease inhibitor cocktail) or 1% IGEPAL CA-630 buffer (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 7.4, and Roche cOmplete protease inhibitor cocktail). .. After incubation, beads were thrice washed with ice-cold PBS at the experimental pH, eluted by heating at 95°C with 2× Laemmli buffer for 10 min and analyzed by Western blotting.

Article Title: Loss of Tyrosine Phosphatase Dependent Inhibition Promotes Activation of Tyrosine Kinase c-Src in Detached Pancreatic Cells
Article Snippet: Paragraph title: Western Blot Analysis ... Adherent cells were washed twice with ice-cold tris-buffered saline (TBS), followed by lysis for 10 min in NP-40 buffer (150 mM NaCl, 5 mM EDTA, 1% NP-40, pH 7.4), supplemented with one tablet complete mini-EDTA-free protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany), and sodium orthovanadate (1 mM, pH 7.4).

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol). .. Protein concentration was equilibrated across experimental samples and Western Blot samples were prepared using 2X Sample Buffer (+β-me) and heated to ≥ 95C for ≥ 5 min. NP40 lysates were lysed for 15 min at 4C and then centrifuged at 20,000g for 20 min at 4C, and the protein concentration of the supernatant was quantified using Advanced Protein Assay Reagent (Cytoskeleton, modified Method 4).

Lysis:

Article Title: Loss of Tyrosine Phosphatase Dependent Inhibition Promotes Activation of Tyrosine Kinase c-Src in Detached Pancreatic Cells
Article Snippet: .. Adherent cells were washed twice with ice-cold tris-buffered saline (TBS), followed by lysis for 10 min in NP-40 buffer (150 mM NaCl, 5 mM EDTA, 1% NP-40, pH 7.4), supplemented with one tablet complete mini-EDTA-free protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany), and sodium orthovanadate (1 mM, pH 7.4). .. Cells were scraped from plates, and cell lysates were clarified by centrifugation for 5 min at 13,000 × g and 4 °C.

Article Title: Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer
Article Snippet: For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). .. Precipitates were washed extensively with lysis buffer.

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol). .. Following lysis on ice at 4C, SDS lysates were heated at ≥ 95C for ≥ 5 min and then protein concentration was measured via Pierce BCA assay.

Binding Assay:

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10
Article Snippet: Paragraph title: In Vitro Binding Assay ... THP-1 WT cells were lysed in Buffer L (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 5.5, and Roche cOmplete protease inhibitor cocktail) or 1% IGEPAL CA-630 buffer (50 mM Tris–Cl, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630, pH 7.4, and Roche cOmplete protease inhibitor cocktail).

Article Title: Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer
Article Snippet: For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). .. After centrifugation, the supernatants were precleared with protein A/G beads at 4°C for 2 h. Pre-cleared lysates were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was incubated for 4 h at 4°C.

BIA-KA:

Article Title: Sox11 Reduces Caspase-6 Cleavage and Activity
Article Snippet: Cells were harvested 24hours post-transfection and lysed on ice using an NP-40 buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.02% NaN3 ) supplemented with complete protease inhibitor (Roche). .. Protein concentration was determined by the BCA method (Pierce)

Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma
Article Snippet: Immunoblot Cells were pelleted by centrifugation, washed with ice-cold PBS and lysed on ice for 20 min in modified NP-40 buffer (10mM/1mM KPO4/EDTA pH 7.05, 5mM EGTA pH 7.2, 10mM MgCl2, 50mM glycerophosphate pH 7.2, 0.5% NP-40, 0.1% Brij-35) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 1mM DTT, 1mM Va3VO4 and 1mM PMSF. .. Protein concentrations of whole-cell lysates were determined by BCA protein assays (Pierce), and equivalent protein amounts (20-60 μg) were electrophoresed through 4-12% Bis-Tris acrylamide gels.

Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
Article Snippet: All media were then aspirated, cells were washed once in cold 1X PBS (usually supplemented with phosphatase inhibitors NaF, β-glycerophosphate and sodium orthovanadate) and then lysed either in NP-40 buffer (50mM Tris HCl, 120mM NaCl, 0.5% NP40, 1mM EDTA, 1mM DTT), plus phosphatase inhibitors (NaF, β-glycerophosphate and sodium orthovanadate) and protease inhibitors tablet (Roche cOmplete Mini) or in 2% SDS buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol). .. Following lysis on ice at 4C, SDS lysates were heated at ≥ 95C for ≥ 5 min and then protein concentration was measured via Pierce BCA assay.

SDS Page:

Article Title: Regulation of p53 stability and function by the deubiquitinating enzyme USP42
Article Snippet: Cell extracts were prepared in either NP-40 buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40) or RIPA buffer (50 mM Tris–HCl, pH 8.0, 120 mM NaCl, 1% NP-40, 1% SDS 0.5% DOC, protease inhibitor cocktail (Roche)). .. Proteins were resolved by SDS–PAGE and transferred onto nitrocellulose membrane (Millipore).

Article Title: Kaposi's Sarcoma-Associated Herpesvirus K7 Protein Targets a Ubiquitin-Like/Ubiquitin-Associated Domain-Containing Protein To Promote Protein Degradation
Article Snippet: Cells were harvested and then lysed with NP-40 buffer (0.15 M NaCl, 1% Nonidet P-40, 50 mM Tris, pH 7.5) containing 0.1 mM Na2 VO3 , 1 mM NaF, and protease inhibitor cocktail (Roche, Mannheim, Germany). .. For immunoblotting, polypeptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad).

Software:

Article Title: Regulation of p53 stability and function by the deubiquitinating enzyme USP42
Article Snippet: Cell extracts were prepared in either NP-40 buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40) or RIPA buffer (50 mM Tris–HCl, pH 8.0, 120 mM NaCl, 1% NP-40, 1% SDS 0.5% DOC, protease inhibitor cocktail (Roche)). .. Immunoblots were quantitated using ImageJ software or the Licor Odyssey system.

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  • 94
    Roche te np 40 buffer
    Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods . Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of <t>NP-40</t> detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ , and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.
    Te Np 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/te np 40 buffer/product/Roche
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    te np 40 buffer - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    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
    Price from $9.99 to $1999.99
    nondenaturing np 40 lysis buffer - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    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.
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    Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods . Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of NP-40 detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ , and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.

    Journal: Molecular Biology of the Cell

    Article Title: Cdc48 and ubiquilins confer selective anterograde protein sorting and entry into the multivesicular body in yeast

    doi: 10.1091/mbc.E17-11-0652

    Figure Lengend Snippet: Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods . Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of NP-40 detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ , and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.

    Article Snippet: Next, 25 O.D.600 units were taken for coimmunoprecipitation (co-IP) in 1 ml of TE/NP-40 buffer with 10 μl of monoclonal anti-HA antibody (Roche) by incubation on rotator overnight at 4°C.

    Techniques: Expressing, Sedimentation, Western Blot

    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