Structured Review

Roche protease inhibitor cocktail roche
Protease Inhibitor Cocktail Roche, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: 2.3.2 VWF A3-GST (glutathione S-transferase) preparation A recombinant GST-tagged human VWF-A3 domain plasmid was obtained by cloning the VWF-A3 ORF into the bacterial expression vector pGEX-2T. .. Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka).

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: [ ] SV2A point mutants were synthesized at Genscript and cloned into pcDNA3.1(+) (Life Technologies, Gent, Belgium). .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Article Title: Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
Article Snippet: Human SV2A was cloned from a fetal brain cDNA library as described in (Lynch et al ., ) and stably expressed in HEK cells. .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Paragraph title: Cloning and overexpression of TbrHGPRT ... Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer.

Centrifugation:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: .. Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka). .. Suspensions were sonicated and Triton X-100 was adjusted to 1% (v/v).

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: The pellet was washed once with ice cold PBS using the same centrifugation protocol. .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Article Title: Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
Article Snippet: The pellet was washed once with ice-cold PBS using the same centrifugation protocol. .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer. .. The soluble fraction was separated by centrifugation at 4 °C.

Article Title: Curcumin Modulates ?-Synuclein Aggregation and Toxicity
Article Snippet: Briefly, the IPTG induced bacterial cells were pelleteddown by centrifugation. .. The pellet was resuspended in buffer (50 mMTris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail(Roche), sonicated followed by heating in boiling water bath for 20min.

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche]. .. Supernatants were collected after centrifugation (13000 g for 25 min) and the total protein concentration was measured by the Bradford method (Biorad).

Amplification:

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: For PCR-2, PCR-1-derived ER-β and ER-α were diluted tenfold, and a 3 μL aliquot was amplified with 40 pmol of inner primers as per PCR1 conditions. β-actin cDNA (0.5 μL) served as internal control. .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche].

Stable Transfection:

Article Title: Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
Article Snippet: Human SV2A was cloned from a fetal brain cDNA library as described in (Lynch et al ., ) and stably expressed in HEK cells. .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Synthesized:

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: [ ] SV2A point mutants were synthesized at Genscript and cloned into pcDNA3.1(+) (Life Technologies, Gent, Belgium). .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Construct:

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: Final constructs were verified by sequencing and were expressed using transient transfections in FreeStyleTM 293-F suspension cells using 293fectinTM following the manufacturers instruction (all from Life Technologies). .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Cell colonies containing the construct of interest were selected for growth on agar LB plates supplemented with ampicillin and chloramphenicol. .. Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer.

Incubation:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka). .. Suspensions were incubated at room temperature for 15 min on a roller mixer and centrifuged at 18,000 g for 20 min and supernatants were pooled.

Article Title: Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor α membrane trafficking to promote breast cancer progression
Article Snippet: .. To improve the detection of ADAM17, cells were lysed in TX-100 lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) supplemented with complete protease inhibitor cocktail (Roche) and 10 mM 1,10-phenanthroline to prevent autocatalysis of ADAM17, and centrifuged at 20,000 × g for 20 min. Lysates were then mixed with 50 μL of washed concanavalin A (Con A) Agarose beads and incubated for 2–3 h at 4 °C on a rotor. ..

Article Title: The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres
Article Snippet: Exponentially growing D-Mel2 cells were lysed on ice in lysis buffer (for Polo-GFP cell line: 40 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM EGTA, 1% Triton-X-100, and protease inhibitor cocktail (Roche, UK); for GFP-Aurora B cell line: 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% deoxycholate, and protease inhibitor cocktail -Roche). .. Cell lysates were separately incubated with either mAb anti-GFP or mouse IgG bound to Dynabeads protein G for 1 h at 4°C.

Expressing:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: 2.3.2 VWF A3-GST (glutathione S-transferase) preparation A recombinant GST-tagged human VWF-A3 domain plasmid was obtained by cloning the VWF-A3 ORF into the bacterial expression vector pGEX-2T. .. Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka).

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Induction of expression was at 30 °C for 8 hours upon the addition of 0.5 M isopropyl β-D-1-thiogalactopyranoside (IPTG). .. Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer.

Article Title: Curcumin Modulates ?-Synuclein Aggregation and Toxicity
Article Snippet: For 15 N labeled α-Syn expression, minimal mediacontaining 15 N-labeled ammonium chloride was used as anitrogen source. .. The pellet was resuspended in buffer (50 mMTris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail(Roche), sonicated followed by heating in boiling water bath for 20min.

Western Blot:

Article Title: HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells
Article Snippet: Paragraph title: Western blotting ... The cells were lysed in lysis buffer on ice for 30 minutes (Tris HCl, 20 mM; NaCl, 150 mM; NaF 100 mM; Na4 P2 07 10 mM; EDTA 10 mM with 1% Triton and protease inhibitor cocktail-Roche) and centrifuged at 4°C to remove of the insoluble cell pellets.

Over Expression:

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Paragraph title: Cloning and overexpression of TbrHGPRT ... Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer.

Transfection:

Article Title: Osteoblastic PLEKHO1 contributes to joint inflammation in rheumatoid arthritis
Article Snippet: .. 2.6 Immunoprecipitation and immunoblotting At 36 h after the transfection, HEK293T were lysed in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1mM NaF, 1mM dithiothreitol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10mM NaF and 1mM Na3 VO4 ). .. Immunoprecipitations were performed using anti-Flag or anti-Myc antibody and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C.

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: Final constructs were verified by sequencing and were expressed using transient transfections in FreeStyleTM 293-F suspension cells using 293fectinTM following the manufacturers instruction (all from Life Technologies). .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Chromatography:

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer. .. The recombinant enzyme was purified by Ni2+ chromatography column (ProfinityTM IMAC resin) at 4 °C.

Protease Inhibitor:

Article Title: Osteoblastic PLEKHO1 contributes to joint inflammation in rheumatoid arthritis
Article Snippet: .. 2.6 Immunoprecipitation and immunoblotting At 36 h after the transfection, HEK293T were lysed in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1mM NaF, 1mM dithiothreitol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10mM NaF and 1mM Na3 VO4 ). .. Immunoprecipitations were performed using anti-Flag or anti-Myc antibody and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C.

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: .. Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka). .. Suspensions were sonicated and Triton X-100 was adjusted to 1% (v/v).

Article Title: Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor α membrane trafficking to promote breast cancer progression
Article Snippet: .. To improve the detection of ADAM17, cells were lysed in TX-100 lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) supplemented with complete protease inhibitor cocktail (Roche) and 10 mM 1,10-phenanthroline to prevent autocatalysis of ADAM17, and centrifuged at 20,000 × g for 20 min. Lysates were then mixed with 50 μL of washed concanavalin A (Con A) Agarose beads and incubated for 2–3 h at 4 °C on a rotor. ..

Article Title: Toxic amyloid-β oligomers induced self-replication in astrocytes triggering neuronal injury
Article Snippet: .. Ice-cold Tris-buffered saline (TBS)consisting of 20 mM Tris-HCl, 150 mM NaCl (pH 7.4), 1% Triton X-100, 2% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche) was added to the frozen hemisphere (excluding the cerebellum),which was then homogenized using a mechanical Dounce homogenizer and then centrifuged for 30 min at 13,000 g . .. Aβ oligomers were purified with a Thermo Scientific™ direct immunoprecipitation Kit (Thermo Scientific) according to the manufacturer's instructions.

Article Title: Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
Article Snippet: .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche. ..

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: .. Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer. ..

Article Title: Curcumin Modulates ?-Synuclein Aggregation and Toxicity
Article Snippet: .. The pellet was resuspended in buffer (50 mMTris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail(Roche), sonicated followed by heating in boiling water bath for 20min. ..

Article Title: HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells
Article Snippet: .. The cells were lysed in lysis buffer on ice for 30 minutes (Tris HCl, 20 mM; NaCl, 150 mM; NaF 100 mM; Na4 P2 07 10 mM; EDTA 10 mM with 1% Triton and protease inhibitor cocktail-Roche) and centrifuged at 4°C to remove of the insoluble cell pellets. ..

Article Title: The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres
Article Snippet: .. Exponentially growing D-Mel2 cells were lysed on ice in lysis buffer (for Polo-GFP cell line: 40 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM EGTA, 1% Triton-X-100, and protease inhibitor cocktail (Roche, UK); for GFP-Aurora B cell line: 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% deoxycholate, and protease inhibitor cocktail -Roche). .. Cell lysates were separately incubated with either mAb anti-GFP or mouse IgG bound to Dynabeads protein G for 1 h at 4°C.

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche]. .. Supernatants were collected after centrifugation (13000 g for 25 min) and the total protein concentration was measured by the Bradford method (Biorad).

Protein Concentration:

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche. .. Finally, the pellet was resuspended in a buffer containing 20 mM Tris-HCl (pH 7.4) and 250 mM of sucrose at a protein concentration of 5 to 10 mg ml-1 and stored in liquid nitrogen.

Article Title: Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
Article Snippet: The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche. .. Finally, the pellet was resuspended in buffer A at a protein concentration of 5 to 10 mg·mL−1 and stored in liquid nitrogen.

Polymerase Chain Reaction:

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: After normalization to the fluorescent β-actin PCR band intensity, ER-β and ER-α mRNA were expressed as arbitrary units of fluorescence. .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche].

Sonication:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka). .. Suspensions were sonicated and Triton X-100 was adjusted to 1% (v/v).

Article Title: Curcumin Modulates ?-Synuclein Aggregation and Toxicity
Article Snippet: .. The pellet was resuspended in buffer (50 mMTris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail(Roche), sonicated followed by heating in boiling water bath for 20min. ..

Binding Assay:

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche. .. Competition binding experiments .

Fluorescence:

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: After normalization to the fluorescent β-actin PCR band intensity, ER-β and ER-α mRNA were expressed as arbitrary units of fluorescence. .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche].

Labeling:

Article Title: Curcumin Modulates ?-Synuclein Aggregation and Toxicity
Article Snippet: For 15 N labeled α-Syn expression, minimal mediacontaining 15 N-labeled ammonium chloride was used as anitrogen source. .. The pellet was resuspended in buffer (50 mMTris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail(Roche), sonicated followed by heating in boiling water bath for 20min.

Purification:

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer. .. The recombinant enzyme was purified by Ni2+ chromatography column (ProfinityTM IMAC resin) at 4 °C.

Protein Purification:

Article Title: Curcumin Modulates ?-Synuclein Aggregation and Toxicity
Article Snippet: Paragraph title: Protein Purification ... The pellet was resuspended in buffer (50 mMTris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail(Roche), sonicated followed by heating in boiling water bath for 20min.

Sequencing:

Article Title: Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis
Article Snippet: Paragraph title: Sequence Analysis and Model Building ... The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

Lysis:

Article Title: Osteoblastic PLEKHO1 contributes to joint inflammation in rheumatoid arthritis
Article Snippet: .. 2.6 Immunoprecipitation and immunoblotting At 36 h after the transfection, HEK293T were lysed in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1mM NaF, 1mM dithiothreitol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10mM NaF and 1mM Na3 VO4 ). .. Immunoprecipitations were performed using anti-Flag or anti-Myc antibody and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C.

Article Title: Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor α membrane trafficking to promote breast cancer progression
Article Snippet: .. To improve the detection of ADAM17, cells were lysed in TX-100 lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) supplemented with complete protease inhibitor cocktail (Roche) and 10 mM 1,10-phenanthroline to prevent autocatalysis of ADAM17, and centrifuged at 20,000 × g for 20 min. Lysates were then mixed with 50 μL of washed concanavalin A (Con A) Agarose beads and incubated for 2–3 h at 4 °C on a rotor. ..

Article Title: HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells
Article Snippet: .. The cells were lysed in lysis buffer on ice for 30 minutes (Tris HCl, 20 mM; NaCl, 150 mM; NaF 100 mM; Na4 P2 07 10 mM; EDTA 10 mM with 1% Triton and protease inhibitor cocktail-Roche) and centrifuged at 4°C to remove of the insoluble cell pellets. ..

Article Title: The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres
Article Snippet: .. Exponentially growing D-Mel2 cells were lysed on ice in lysis buffer (for Polo-GFP cell line: 40 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM EGTA, 1% Triton-X-100, and protease inhibitor cocktail (Roche, UK); for GFP-Aurora B cell line: 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% deoxycholate, and protease inhibitor cocktail -Roche). .. Cell lysates were separately incubated with either mAb anti-GFP or mouse IgG bound to Dynabeads protein G for 1 h at 4°C.

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche]. .. Supernatants were collected after centrifugation (13000 g for 25 min) and the total protein concentration was measured by the Bradford method (Biorad).

cDNA Library Assay:

Article Title: Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
Article Snippet: Human SV2A was cloned from a fetal brain cDNA library as described in (Lynch et al ., ) and stably expressed in HEK cells. .. The resulting pellet was homogenized in a buffer containing 15 mM Tris-HCl, 1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2 (pH 7.5) supplemented with complete protease inhibitor cocktail Roche.

SDS Page:

Article Title: The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres
Article Snippet: Exponentially growing D-Mel2 cells were lysed on ice in lysis buffer (for Polo-GFP cell line: 40 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM EGTA, 1% Triton-X-100, and protease inhibitor cocktail (Roche, UK); for GFP-Aurora B cell line: 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% deoxycholate, and protease inhibitor cocktail -Roche). .. All samples were subjected to SDS-PAGE and analyzed by immunoblotting as described before.

Plasmid Preparation:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: 2.3.2 VWF A3-GST (glutathione S-transferase) preparation A recombinant GST-tagged human VWF-A3 domain plasmid was obtained by cloning the VWF-A3 ORF into the bacterial expression vector pGEX-2T. .. Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka).

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: The plasmid containing this gene was then used to transform competent BL-21 (DE3) E. coli cells. .. Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer.

Recombinant:

Article Title: Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF
Article Snippet: 2.3.2 VWF A3-GST (glutathione S-transferase) preparation A recombinant GST-tagged human VWF-A3 domain plasmid was obtained by cloning the VWF-A3 ORF into the bacterial expression vector pGEX-2T. .. Cells were harvested by centrifugation at 4500g for 20 min, and pellets were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka).

Article Title: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase
Article Snippet: Cells were lysed by the addition of lysozyme (2–4 mg per ml of cells) in the presence of protease inhibitor cocktail Roche® (1 tablet per 50 mL of cells) and DNase 50 μg/ml of cells in buffer. .. The recombinant enzyme was purified by Ni2+ chromatography column (ProfinityTM IMAC resin) at 4 °C.

Agarose Gel Electrophoresis:

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: DNA fragments were separated on a 2% agarose gel stained with ethidium bromide and size assessed by comparison with 100 bp DNA marker using Molecular Imager ChemiDOC XRS+ (Biorad). .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche].

Immunoprecipitation:

Article Title: Osteoblastic PLEKHO1 contributes to joint inflammation in rheumatoid arthritis
Article Snippet: .. 2.6 Immunoprecipitation and immunoblotting At 36 h after the transfection, HEK293T were lysed in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1mM NaF, 1mM dithiothreitol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10mM NaF and 1mM Na3 VO4 ). .. Immunoprecipitations were performed using anti-Flag or anti-Myc antibody and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C.

Article Title: The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres
Article Snippet: Paragraph title: Immunoprecipitation ... Exponentially growing D-Mel2 cells were lysed on ice in lysis buffer (for Polo-GFP cell line: 40 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM EGTA, 1% Triton-X-100, and protease inhibitor cocktail (Roche, UK); for GFP-Aurora B cell line: 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% deoxycholate, and protease inhibitor cocktail -Roche).

Marker:

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: DNA fragments were separated on a 2% agarose gel stained with ethidium bromide and size assessed by comparison with 100 bp DNA marker using Molecular Imager ChemiDOC XRS+ (Biorad). .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche].

Staining:

Article Title: Phytoestrogens/insoluble fibers and colonic estrogen receptor ?: Randomized, double-blind, placebo-controlled study
Article Snippet: DNA fragments were separated on a 2% agarose gel stained with ethidium bromide and size assessed by comparison with 100 bp DNA marker using Molecular Imager ChemiDOC XRS+ (Biorad). .. For the extraction of proteins, colonic biopsies were homogenized in lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 4 mmol/L EDTA, 2% NP40, 0.5% Na deoxycholate, 1 mmol/L sodium orthovanadate and a protease inhibitor cocktail-Roche].

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  • 96
    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: 96/100, 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
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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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    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