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Roche protease inhibitor cocktail
Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 8944 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Centrifugation:

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: .. Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Twenty-five milligrams of brain Triton X-100 extract were flowed 10 times through columns loaded with 500 μ g of each GST fusion bound to Glutathione Sepharose Fast Flow beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

BIA-KA:

Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
Article Snippet: For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland). .. For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland).

Article Title: A role for miR-132 in learned safety
Article Snippet: Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)). .. Cell/tissue homogenate was sonicated and total protein content was quantified using a commercial kit (PierceTM BCA Protein Assay Kit, Thermoscientific) with assistance of the software GEN5 (BioTek, Vermont, USA).

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: .. Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Twenty-five milligrams of brain Triton X-100 extract were flowed 10 times through columns loaded with 500 μ g of each GST fusion bound to Glutathione Sepharose Fast Flow beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

Article Title: Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners
Article Snippet: Whole cell lysates were collected 48–72 hr after transfection using RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with addition of protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktails (Sigma). .. Total protein concentration was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific).

Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner
Article Snippet: .. Western blot Whole cell extracts were prepared in 50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 10 mM NaF, 10% Glycerol, supplemented with protease inhibitor cocktail (Roche, Cat. No. 04693116001) and quantitated using a BCA approach. ..

Construct:

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: .. YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Incubation:

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Approximately 50 μg of protein extract was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to nitrocellulose membrane (Sigma) and incubated with specific antibodies.

Article Title: Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers
Article Snippet: Immunoblotting Cells were lysed in a buffer containing 10 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail (Roche). .. Extracts were subjected to SDS-polyacrilamyde gel electrophoresis (SDS-PAGE), transferred to PVDF membrane (Invitrogen), and immunoblotted with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Article Title: Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development
Article Snippet: In brief, 3 × 106 cells were incubated with 10 μg/mL cycloheximide (Sigma #01810) for the indicated time and washed twice with PBS×1. .. Cell extracts were prepared in 100 μL RIPA buffer supplemented with a protease inhibitors cocktail (Roche #11873580) and 120 μg of cell extract was resolved on SDS-PAGE.

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Protein Misfolding Cyclic Amplification:

Article Title: Recombinant prion protein induces a new transmissible prion disease in wild-type animals
Article Snippet: Paragraph title: Protein misfolding cyclic amplification ... The composition of conversion buffer was as previously described [ ]: Ca2+ - and Mg2+ -free PBS, pH 7.5, supplemented with 0.15 M NaCl, 1.0% Triton, and 1 tablet of complete protease inhibitors cocktail (Roche, Cat# 1836145) per 50 ml of conversion buffer.

Expressing:

Article Title: Expression and Splice Variant Analysis of Human TCF4 Transcription Factor in Esophageal Cancer
Article Snippet: Tissues or cell pellets were homogenized in ice-cold RIPA buffer containing complete protease inhibitors cocktail (Roche (China) Ltd., Shanghai, China). .. The expression of TCF4 protein was normalized by that of GAPDH.

Bradford Assay:

Article Title: Expression and Splice Variant Analysis of Human TCF4 Transcription Factor in Esophageal Cancer
Article Snippet: Tissues or cell pellets were homogenized in ice-cold RIPA buffer containing complete protease inhibitors cocktail (Roche (China) Ltd., Shanghai, China). .. The extraction mixture was then centrifuged at 12000g at 4℃ for 20 min and the protein concentration in the supernatant was determined by Bradford assay.

Western Blot:

Article Title: IC-2 Suppresses Proliferation and Induces Apoptosis of Bladder Cancer Cells via the Wnt/β-Catenin Pathway
Article Snippet: Paragraph title: Western blot assay ... Bladder cancer cells were lysed using protein extraction reagent radio-immunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) with protease inhibitors cocktail and phenylmethanesulfonyl fluoride (PMSF) (Roche, Switzerland).

Article Title: Expression and Splice Variant Analysis of Human TCF4 Transcription Factor in Esophageal Cancer
Article Snippet: Paragraph title: Proteins samples and Western blotting ... Tissues or cell pellets were homogenized in ice-cold RIPA buffer containing complete protease inhibitors cocktail (Roche (China) Ltd., Shanghai, China).

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: .. Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Protein concentration was measured using the Bio-Rad protein assay kit.

Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
Article Snippet: Paragraph title: 4.5. Protein Isolation and Western Blotting ... For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland).

Article Title: Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers
Article Snippet: Immunoblotting Cells were lysed in a buffer containing 10 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail (Roche). .. Western blot densitometry was performed using ImageJ software (US National Institutes of Health).

Article Title: Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells
Article Snippet: .. Fractionation of nuclear proteins and Western blotting Cell pellets were resuspended in a hypotonic buffer (10 mM HEPES-K+ , pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 0.5 M dithiothreitol) in the presence of a protease inhibitors cocktail (Roche). ..

Article Title: Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro
Article Snippet: Paragraph title: Western blot analysis ... Cells were harvested in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton N-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) 48 h after siRNA transfection.

Article Title: A role for miR-132 in learned safety
Article Snippet: Paragraph title: Protein isolation and Western Blot ... Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)).

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Immunoprecipitates were washed three times with RIPA buffer lacking SDS, eluted with Laemmli Sample Buffer (BioRad, Hercules, CA) by incubation at 95°C for 5 min and resolved by standard SDS-PAGE gel electrophoresis and Western Blotting.

Article Title: E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis
Article Snippet: Paragraph title: Western blotting ... The lysis buffer was added with protease inhibitors cocktail (1:100, Roche).

Article Title: Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners
Article Snippet: Paragraph title: Cell culture, transient transfection and western blotting ... Whole cell lysates were collected 48–72 hr after transfection using RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with addition of protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktails (Sigma).

Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner
Article Snippet: .. Western blot Whole cell extracts were prepared in 50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 10 mM NaF, 10% Glycerol, supplemented with protease inhibitor cocktail (Roche, Cat. No. 04693116001) and quantitated using a BCA approach. ..

Derivative Assay:

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: .. YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Transfection:

Article Title: Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro
Article Snippet: .. Cells were harvested in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton N-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) 48 h after siRNA transfection. .. The protein concentration was quantified using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Article Title: Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development
Article Snippet: Plasmid transfection was performed using FugeneHD® reagent. .. Cell extracts were prepared in 100 μL RIPA buffer supplemented with a protease inhibitors cocktail (Roche #11873580) and 120 μg of cell extract was resolved on SDS-PAGE.

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: .. YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Article Title: Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners
Article Snippet: .. Whole cell lysates were collected 48–72 hr after transfection using RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with addition of protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktails (Sigma). .. Total protein concentration was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific).

Concentration Assay:

Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
Article Snippet: For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland). .. Protein samples were then diluted to a final concentration 2 μg/μL with 2× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and stored at −20 °C until analysis.

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: .. Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Twenty-five milligrams of brain Triton X-100 extract were flowed 10 times through columns loaded with 500 μ g of each GST fusion bound to Glutathione Sepharose Fast Flow beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: .. YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Protease Inhibitor:

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: .. Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Protein concentration was measured using the Bio-Rad protein assay kit.

Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
Article Snippet: .. For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland). .. Protein concentrations were determined using a BCA protein assay (ThermoFisher Scientific, Waltham, MA, USA).

Article Title: Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro
Article Snippet: .. Cells were harvested in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton N-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) 48 h after siRNA transfection. .. The protein concentration was quantified using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Immunoprecipitations were performed from cell lysates generated in lysis buffer (20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 1% Triton X-100) in the presence of protease inhibitor cocktail (Roche) and 1 mM orthovanadate.

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: .. YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner
Article Snippet: .. Western blot Whole cell extracts were prepared in 50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 10 mM NaF, 10% Glycerol, supplemented with protease inhibitor cocktail (Roche, Cat. No. 04693116001) and quantitated using a BCA approach. ..

Cell Culture:

Article Title: Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners
Article Snippet: Paragraph title: Cell culture, transient transfection and western blotting ... Whole cell lysates were collected 48–72 hr after transfection using RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with addition of protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktails (Sigma).

Generated:

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Immunoprecipitations were performed from cell lysates generated in lysis buffer (20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 1% Triton X-100) in the presence of protease inhibitor cocktail (Roche) and 1 mM orthovanadate.

Protein Concentration:

Article Title: Expression and Splice Variant Analysis of Human TCF4 Transcription Factor in Esophageal Cancer
Article Snippet: Tissues or cell pellets were homogenized in ice-cold RIPA buffer containing complete protease inhibitors cocktail (Roche (China) Ltd., Shanghai, China). .. The extraction mixture was then centrifuged at 12000g at 4℃ for 20 min and the protein concentration in the supernatant was determined by Bradford assay.

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Protein concentration was measured using the Bio-Rad protein assay kit.

Article Title: Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro
Article Snippet: Cells were harvested in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton N-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) 48 h after siRNA transfection. .. The protein concentration was quantified using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: .. Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Twenty-five milligrams of brain Triton X-100 extract were flowed 10 times through columns loaded with 500 μ g of each GST fusion bound to Glutathione Sepharose Fast Flow beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

Article Title: E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis
Article Snippet: The lysis buffer was added with protease inhibitors cocktail (1:100, Roche). .. The protein concentration was quantified using Bradford Protein Assay Kit (Beyotime, P0006).

Article Title: Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners
Article Snippet: Whole cell lysates were collected 48–72 hr after transfection using RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with addition of protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktails (Sigma). .. Total protein concentration was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific).

Polymerase Chain Reaction:

Article Title: Recombinant prion protein induces a new transmissible prion disease in wild-type animals
Article Snippet: The composition of conversion buffer was as previously described [ ]: Ca2+ - and Mg2+ -free PBS, pH 7.5, supplemented with 0.15 M NaCl, 1.0% Triton, and 1 tablet of complete protease inhibitors cocktail (Roche, Cat# 1836145) per 50 ml of conversion buffer. .. Samples in 0.2 ml thin-wall PCR tubes were placed in a floating rack inside Misonix-3000 cup-horn sonicator, filled with 300 ml water.

Sonication:

Article Title: A role for miR-132 in learned safety
Article Snippet: Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)). .. Cell/tissue homogenate was sonicated and total protein content was quantified using a commercial kit (PierceTM BCA Protein Assay Kit, Thermoscientific) with assistance of the software GEN5 (BioTek, Vermont, USA).

DC Protein Assay:

Article Title: Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro
Article Snippet: Cells were harvested in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton N-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) 48 h after siRNA transfection. .. The protein concentration was quantified using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Nucleic Acid Electrophoresis:

Article Title: IC-2 Suppresses Proliferation and Induces Apoptosis of Bladder Cancer Cells via the Wnt/β-Catenin Pathway
Article Snippet: Bladder cancer cells were lysed using protein extraction reagent radio-immunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) with protease inhibitors cocktail and phenylmethanesulfonyl fluoride (PMSF) (Roche, Switzerland). .. Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes.

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Approximately 50 μg of protein extract was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to nitrocellulose membrane (Sigma) and incubated with specific antibodies.

Article Title: Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers
Article Snippet: Immunoblotting Cells were lysed in a buffer containing 10 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail (Roche). .. Extracts were subjected to SDS-polyacrilamyde gel electrophoresis (SDS-PAGE), transferred to PVDF membrane (Invitrogen), and immunoblotted with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Immunoprecipitates were washed three times with RIPA buffer lacking SDS, eluted with Laemmli Sample Buffer (BioRad, Hercules, CA) by incubation at 95°C for 5 min and resolved by standard SDS-PAGE gel electrophoresis and Western Blotting.

Isolation:

Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
Article Snippet: .. For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland). .. Protein concentrations were determined using a BCA protein assay (ThermoFisher Scientific, Waltham, MA, USA).

Article Title: A role for miR-132 in learned safety
Article Snippet: Paragraph title: Protein isolation and Western Blot ... Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)).

Flow Cytometry:

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Twenty-five milligrams of brain Triton X-100 extract were flowed 10 times through columns loaded with 500 μ g of each GST fusion bound to Glutathione Sepharose Fast Flow beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

Purification:

Article Title: A role for miR-132 in learned safety
Article Snippet: Protein isolation and Western Blot Proteins were purified from 3t3 cells and BLA tissue. .. Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)).

Protein Extraction:

Article Title: IC-2 Suppresses Proliferation and Induces Apoptosis of Bladder Cancer Cells via the Wnt/β-Catenin Pathway
Article Snippet: .. Bladder cancer cells were lysed using protein extraction reagent radio-immunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) with protease inhibitors cocktail and phenylmethanesulfonyl fluoride (PMSF) (Roche, Switzerland). .. Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes.

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: .. Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Protein concentration was measured using the Bio-Rad protein assay kit.

Article Title: E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis
Article Snippet: The total protein samples of the heart tissues were extracted using Cell Lysis and Protein Extraction Kit (Solarbio, BC3640) according to manufacturer’s instructions. .. The lysis buffer was added with protease inhibitors cocktail (1:100, Roche).

Bradford Protein Assay:

Article Title: E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis
Article Snippet: The lysis buffer was added with protease inhibitors cocktail (1:100, Roche). .. The protein concentration was quantified using Bradford Protein Assay Kit (Beyotime, P0006).

SDS Page:

Article Title: IC-2 Suppresses Proliferation and Induces Apoptosis of Bladder Cancer Cells via the Wnt/β-Catenin Pathway
Article Snippet: Bladder cancer cells were lysed using protein extraction reagent radio-immunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) with protease inhibitors cocktail and phenylmethanesulfonyl fluoride (PMSF) (Roche, Switzerland). .. Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes.

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. Approximately 50 μg of protein extract was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to nitrocellulose membrane (Sigma) and incubated with specific antibodies.

Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
Article Snippet: For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions with the addition of a protease inhibitors cocktail (Protease Inhibitor cOmplete Mini, Roche, Basel, Switzerland). .. Twenty micrograms (20 μg) of protein lysates were separated in 1.5 mm thick 12% SDS-PAGE gels.

Article Title: Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers
Article Snippet: Immunoblotting Cells were lysed in a buffer containing 10 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail (Roche). .. Extracts were subjected to SDS-polyacrilamyde gel electrophoresis (SDS-PAGE), transferred to PVDF membrane (Invitrogen), and immunoblotted with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Article Title: Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development
Article Snippet: .. Cell extracts were prepared in 100 μL RIPA buffer supplemented with a protease inhibitors cocktail (Roche #11873580) and 120 μg of cell extract was resolved on SDS-PAGE. ..

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Immunoprecipitates were washed three times with RIPA buffer lacking SDS, eluted with Laemmli Sample Buffer (BioRad, Hercules, CA) by incubation at 95°C for 5 min and resolved by standard SDS-PAGE gel electrophoresis and Western Blotting.

Plasmid Preparation:

Article Title: Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development
Article Snippet: Plasmid transfection was performed using FugeneHD® reagent. .. Cell extracts were prepared in 100 μL RIPA buffer supplemented with a protease inhibitors cocktail (Roche #11873580) and 120 μg of cell extract was resolved on SDS-PAGE.

Software:

Article Title: Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15
Article Snippet: Western blot analysis and antibodies The cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). .. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity one software; Bio-Rad, Hercules, CA, USA).

Article Title: Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers
Article Snippet: Immunoblotting Cells were lysed in a buffer containing 10 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail (Roche). .. Western blot densitometry was performed using ImageJ software (US National Institutes of Health).

Article Title: A role for miR-132 in learned safety
Article Snippet: Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)). .. Cell/tissue homogenate was sonicated and total protein content was quantified using a commercial kit (PierceTM BCA Protein Assay Kit, Thermoscientific) with assistance of the software GEN5 (BioTek, Vermont, USA).

Radio Immunoprecipitation:

Article Title: IC-2 Suppresses Proliferation and Induces Apoptosis of Bladder Cancer Cells via the Wnt/β-Catenin Pathway
Article Snippet: .. Bladder cancer cells were lysed using protein extraction reagent radio-immunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) with protease inhibitors cocktail and phenylmethanesulfonyl fluoride (PMSF) (Roche, Switzerland). .. Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes.

Homogenization:

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: Biochemical miscellaneous procedures Tissue homogenization and GST pull-down experiments were carried out as previously described. .. Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA).

Immunoprecipitation:

Article Title: Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD
Article Snippet: .. YAP/TEAD Immunoprecipitation (IP) For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. .. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C.

Fractionation:

Article Title: Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells
Article Snippet: .. Fractionation of nuclear proteins and Western blotting Cell pellets were resuspended in a hypotonic buffer (10 mM HEPES-K+ , pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 0.5 M dithiothreitol) in the presence of a protease inhibitors cocktail (Roche). ..

Lysis:

Article Title: Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells
Article Snippet: Fractionation of nuclear proteins and Western blotting Cell pellets were resuspended in a hypotonic buffer (10 mM HEPES-K+ , pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 0.5 M dithiothreitol) in the presence of a protease inhibitors cocktail (Roche). .. The nuclear pellets were washed twice with hypotonic buffer, and then resuspended in nucleus lysis buffer (20 mM HEPES-K+ , pH 7.9, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2 , 0.5 M dithiothreitol, 25% glycerol) with protease inhibitors.

Article Title: Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro
Article Snippet: .. Cells were harvested in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton N-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) 48 h after siRNA transfection. .. The protein concentration was quantified using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Article Title: A role for miR-132 in learned safety
Article Snippet: .. Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)). .. Cell/tissue homogenate was sonicated and total protein content was quantified using a commercial kit (PierceTM BCA Protein Assay Kit, Thermoscientific) with assistance of the software GEN5 (BioTek, Vermont, USA).

Article Title: Pur-alpha functionally interacts with FUS carrying ALS-associated mutations
Article Snippet: Briefly, four mouse brains were harvested in 20 ml of HB (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and protease inhibitors cocktail (Roche, Mannheim, Germany), homogenized, and the homogenate was spin at 3000 g for 5 min. Triton X-100 to the final concentration of 1% was added to the supernatant and the extract was incubate at 4 °C under constant rotation for 1 h. After centrifugation at 100 000 g for 45 min at 4 °C we collected the supernatant and measured protein concentration with BCA PROTEIN ASSAY KIT (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). .. Immunoprecipitations were performed from cell lysates generated in lysis buffer (20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 1% Triton X-100) in the presence of protease inhibitor cocktail (Roche) and 1 mM orthovanadate.

Article Title: E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis
Article Snippet: .. The lysis buffer was added with protease inhibitors cocktail (1:100, Roche). ..

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  • 97
    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche complete mini protease inhibitor cocktail
    Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with <t>cOmplete</t> Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Complete Mini Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete mini protease inhibitor cocktail/product/Roche
    Average 99 stars, based on 350 article reviews
    Price from $9.99 to $1999.99
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    94
    Roche pbs containing protease inhibitor cocktail
    ST398 CA-MRSA virulence in a mouse model. The seven ST398 CA-MRSA isolates were compared with closely related ST398 MSSA, all ST398 LA-MSSA obtained for this study, and representative Chinese ST5 and ST239 HA-MRSA strains by in vivo analysis of virulence. In addition, two USA300 CA-MRSA strains (SF8300, LAC) and the genome-sequenced LA-MRSA S0385 standard strain were used in the comparisons. Isolate selection for the MSSA and HA-MRSA groups was performed, as in a previous study [ 26 ], by selecting isolates whose average PSM production was similar to that of the entire group and thus reflects the average virulence potential. Notably, for the in vivo infection studies, the ST398 CA-MRSA, MSSA, and ST5/ST239 HA-MRSA isolates were obtained from the corresponding type of human infection (respiratory and skin infection, respectively). See Additional file 1 : Table S2 for isolates that were compared and their characteristics. a Mouse skin infection model. Mice received 10 7 live S. aureus or <t>PBS</t> alone in the right flank by subcutaneous injection. Skin abscesses (length × width) were measured 48 h after infection. b Mouse lung infection model. We pipetted 4 × 10 9 CFU/40 μl S. aureus into the nares of the anesthetized mice. All mice were euthanized 48 h after inoculation and CFU in the lung tissue were determined. a , b Three mice were infected with each strain; the shown symbols represent the average value obtained from three mice. c – f Inflammatory <t>cytokine</t> expression in ST398 CA-MRSA isolates in comparison. The inflammatory cytokines TNF-α and IL-6 were determined in skin ( c , d ) or lung tissue ( e , f ). a – f Statistical analysis is by one-way ANOVA with Dunnett’s post test versus the ST398 CA-MRSA group. * p
    Pbs Containing Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    Image Search Results


    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Expressing

    A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Staining, Immunoprecipitation, Mass Spectrometry, SDS Page, Binding Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Transfection, Transduction, Construct

    Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Journal: Breast Cancer Research : BCR

    Article Title: Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer

    doi: 10.1186/s13058-016-0676-6

    Figure Lengend Snippet: Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Article Snippet: WCL were prepared by adding equal volumes of 2× RIPA buffer supplemented with 25 U of Benzonase Nuclease (Sigma-Aldrich) and cOmplete Mini Protease Inhibitor Cocktail (Roche), incubating on ice for 30 minutes and sonicating for 15 minutes with 30-second on-off intervals.

    Techniques: Expressing, Radio Immunoprecipitation, Protease Inhibitor

    ST398 CA-MRSA virulence in a mouse model. The seven ST398 CA-MRSA isolates were compared with closely related ST398 MSSA, all ST398 LA-MSSA obtained for this study, and representative Chinese ST5 and ST239 HA-MRSA strains by in vivo analysis of virulence. In addition, two USA300 CA-MRSA strains (SF8300, LAC) and the genome-sequenced LA-MRSA S0385 standard strain were used in the comparisons. Isolate selection for the MSSA and HA-MRSA groups was performed, as in a previous study [ 26 ], by selecting isolates whose average PSM production was similar to that of the entire group and thus reflects the average virulence potential. Notably, for the in vivo infection studies, the ST398 CA-MRSA, MSSA, and ST5/ST239 HA-MRSA isolates were obtained from the corresponding type of human infection (respiratory and skin infection, respectively). See Additional file 1 : Table S2 for isolates that were compared and their characteristics. a Mouse skin infection model. Mice received 10 7 live S. aureus or PBS alone in the right flank by subcutaneous injection. Skin abscesses (length × width) were measured 48 h after infection. b Mouse lung infection model. We pipetted 4 × 10 9 CFU/40 μl S. aureus into the nares of the anesthetized mice. All mice were euthanized 48 h after inoculation and CFU in the lung tissue were determined. a , b Three mice were infected with each strain; the shown symbols represent the average value obtained from three mice. c – f Inflammatory cytokine expression in ST398 CA-MRSA isolates in comparison. The inflammatory cytokines TNF-α and IL-6 were determined in skin ( c , d ) or lung tissue ( e , f ). a – f Statistical analysis is by one-way ANOVA with Dunnett’s post test versus the ST398 CA-MRSA group. * p

    Journal: Genome Medicine

    Article Title: Detection and analysis of methicillin-resistant human-adapted sequence type 398 allows insight into community-associated methicillin-resistant Staphylococcus aureus evolution

    doi: 10.1186/s13073-018-0514-9

    Figure Lengend Snippet: ST398 CA-MRSA virulence in a mouse model. The seven ST398 CA-MRSA isolates were compared with closely related ST398 MSSA, all ST398 LA-MSSA obtained for this study, and representative Chinese ST5 and ST239 HA-MRSA strains by in vivo analysis of virulence. In addition, two USA300 CA-MRSA strains (SF8300, LAC) and the genome-sequenced LA-MRSA S0385 standard strain were used in the comparisons. Isolate selection for the MSSA and HA-MRSA groups was performed, as in a previous study [ 26 ], by selecting isolates whose average PSM production was similar to that of the entire group and thus reflects the average virulence potential. Notably, for the in vivo infection studies, the ST398 CA-MRSA, MSSA, and ST5/ST239 HA-MRSA isolates were obtained from the corresponding type of human infection (respiratory and skin infection, respectively). See Additional file 1 : Table S2 for isolates that were compared and their characteristics. a Mouse skin infection model. Mice received 10 7 live S. aureus or PBS alone in the right flank by subcutaneous injection. Skin abscesses (length × width) were measured 48 h after infection. b Mouse lung infection model. We pipetted 4 × 10 9 CFU/40 μl S. aureus into the nares of the anesthetized mice. All mice were euthanized 48 h after inoculation and CFU in the lung tissue were determined. a , b Three mice were infected with each strain; the shown symbols represent the average value obtained from three mice. c – f Inflammatory cytokine expression in ST398 CA-MRSA isolates in comparison. The inflammatory cytokines TNF-α and IL-6 were determined in skin ( c , d ) or lung tissue ( e , f ). a – f Statistical analysis is by one-way ANOVA with Dunnett’s post test versus the ST398 CA-MRSA group. * p

    Article Snippet: Length (L) and width (W) values were used to calculate the area of lesions with the formula L × W. Mouse skin tissue of the same size was taken from the abscess and PBS control groups and homogenated with glass beads with PBS-containing protease inhibitor cocktail (Roche) for cytokine detection.

    Techniques: In Vivo, Selection, Infection, Mouse Assay, Injection, Expressing