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Roche mammalian protease inhibitors
Mammalian Protease Inhibitors, supplied by Roche, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mammalian protease inhibitors/product/Roche
Average 80 stars, based on 1 article reviews
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mammalian protease inhibitors - by Bioz Stars, 2020-03
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Stable Transfection:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ). ..

Enzyme-linked Immunosorbent Assay:

Article Title: IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis
Article Snippet: The apical lobe of each lung was resected and immediately homogenized in NP40 lysis buffer (50 mM Tris [pH 7.4], 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3 VO4 ,1% NP40,0.02% NaN3 ) containing a cocktail of mammalian protease inhibitors (Roche). .. For ELISA determination of lung IL12p40 concentration, we used the BD OptEIA Mouse IL-12 (p40) ELISA Set system per the manufacturer's instructions (BD Biosciences).

Incubation:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ). .. Following incubation for 1 h at room temperature, reaction mixtures were layered onto 1-ml 70% sucrose cushions and centrifuged at 45,000 rpm (90,700 × g ) for 1 h at 4°C in a Beckman TLA-55 rotor.

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: Comparable amounts of beads were incubated with 1 mg cell lysates from 3×Flag-GPR177 stable HeLa cells for 1 h at 4°C. .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis.

Expressing:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ). ..

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: To check GST-protein expression, a portion of GST-protein-conjugated beads was denatured in 4× LDS (Life Technologies, NP0007) at 70°C for 15 min and subjected to SDS-PAGE followed by Coomassie Blue staining (Invitrogen, LC6060). .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis.

Modification:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: A decrease in SUMO-2/3 conjugation of high molecular weight proteins was evident in the cortex of transgenic mice, and although changes in the modification of multiple SUMO-1 and SUMO-2/3 conjugates were apparent in all brain regions, no other significant changes in conjugation or in levels of SUMO conjugation machinery were observed between transgenic and control mice. .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich).

Article Title: DNA Methylation Has a Local Effect on Transcription and Histone Acetylation
Article Snippet: Chromatin immunoprecipitation (ChIP) assays were performed by a modification of the protocol described by Braunstein et al. ( ). .. Approximately 3 × 106 fixed cells were resuspended in 1 ml of radioimmunoprecipitation buffer (10 mM sodium phosphate [pH 7.2], 2 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.2 mM Na3 VO4 , 1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate) that contained mammalian protease inhibitors (Roche).

Article Title: DNA Methylation Dictates Histone H3K4 Methylation ▿
Article Snippet: ChIP assays were performed by a modification of the protocol described by Braunstein et al. ( ) and as described previously ( ) with minor modifications. .. Approximately 5 × 106 fixed cells were resuspended in 1 ml radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) that contained mammalian protease inhibitors (Roche).

Western Blot:

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis. .. β-galactosidase staining, confocal immunofluorescence, immunohistochemistry, RNA in situ hybridization and quantitative RT-PCR For β-galactosidase staining, tissue sections or whole-mount organoids in chamber slides were rinsed with 1× PBS, fixed in fixative (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2 , 5 mM EGTA and 0.02% NP40) for 15 min, washed in PBS three times at room temperature, and stained with staining solution comprising 5 mM K3 Fe(CN)6 , 5 mM K4 Fe(CN)6 , 2 mM MgCl2 , 0.01% sodium deoxycholate, 0.02% NP40, 1 mg/ml X-Gal (Fisher Scientific, 50-213-181) overnight at 37°C.

Conjugation Assay:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: A decrease in SUMO-2/3 conjugation of high molecular weight proteins was evident in the cortex of transgenic mice, and although changes in the modification of multiple SUMO-1 and SUMO-2/3 conjugates were apparent in all brain regions, no other significant changes in conjugation or in levels of SUMO conjugation machinery were observed between transgenic and control mice. .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich).

Transfection:

Article Title: DNA Methylation Has a Local Effect on Transcription and Histone Acetylation
Article Snippet: Exponentially growing cultures of 293/EBNA1 cells transfected with patch-methylated stable episomes were fixed with 1% formaldehyde for 10 min at 25°C. .. Approximately 3 × 106 fixed cells were resuspended in 1 ml of radioimmunoprecipitation buffer (10 mM sodium phosphate [pH 7.2], 2 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.2 mM Na3 VO4 , 1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate) that contained mammalian protease inhibitors (Roche).

Article Title: DNA Methylation Dictates Histone H3K4 Methylation ▿
Article Snippet: Exponentially growing cultures of 293/EBNA1 cells transfected with patch-methylated stable episomes were fixed with 1% formaldehyde for 10 min at room temperature. .. Approximately 5 × 106 fixed cells were resuspended in 1 ml radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) that contained mammalian protease inhibitors (Roche).

Chromatography:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: Recombinant A14C/E45C and A14C/E45C/G89V CA proteins were expressed in Escherichia coli and purified under reducing conditions by anion-exchange chromatography, to > 98% purity ( ). .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ).

Concentration Assay:

Article Title: IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis
Article Snippet: The apical lobe of each lung was resected and immediately homogenized in NP40 lysis buffer (50 mM Tris [pH 7.4], 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3 VO4 ,1% NP40,0.02% NaN3 ) containing a cocktail of mammalian protease inhibitors (Roche). .. For ELISA determination of lung IL12p40 concentration, we used the BD OptEIA Mouse IL-12 (p40) ELISA Set system per the manufacturer's instructions (BD Biosciences).

Protease Inhibitor:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich). .. For the tissue homogenisation protocol, equivalent amounts of tissue were lysed in equivalent buffer volumes, samples were sonicated for 10 s at 4 °C and total protein concentrations were determined as previously reported .

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ). ..

Cell Culture:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ). ..

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: For GST pull-down, GST, GST-RAB8A, GST-CDC42 and GST-JFC-D1 fusion proteins were expressed in BL21 cells induced by 0.5 mM IPTG and cultured at room temperature for 18 h ( ). .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis.

Protein Concentration:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: CA proteins were coassembled (10-mg/ml total protein concentration; 50-μl volume) into tubular structures at predetermined ratios by dialysis overnight into buffer A (50 mM Tris-HCl [pH 8.0], 1 M NaCl, 20 mM 2-mercaptoethanol). .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ).

Sonication:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich). .. For the tissue homogenisation protocol, equivalent amounts of tissue were lysed in equivalent buffer volumes, samples were sonicated for 10 s at 4 °C and total protein concentrations were determined as previously reported .

Article Title: DNA Methylation Has a Local Effect on Transcription and Histone Acetylation
Article Snippet: Approximately 3 × 106 fixed cells were resuspended in 1 ml of radioimmunoprecipitation buffer (10 mM sodium phosphate [pH 7.2], 2 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.2 mM Na3 VO4 , 1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate) that contained mammalian protease inhibitors (Roche). .. The cell suspension was sonicated 20 times for 10 s each by using a Branson Sonifier 450 (output = 5, constant duty cycle).

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: The bacterial cells were then resuspended in 2 ml 1× PBS with 1% Triton X-100, 0.1 mg/ml lysozyme (Sigma, L6876), 1 mM PMSF and 1× bacterial protease inhibitors (Sigma, P8465), incubated on ice for 30 min followed by sonication. .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis.

Article Title: DNA Methylation Dictates Histone H3K4 Methylation ▿
Article Snippet: Approximately 5 × 106 fixed cells were resuspended in 1 ml radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) that contained mammalian protease inhibitors (Roche). .. The cell suspension was sonicated and centrifuged as described previously ( ).

Recombinant:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: Recombinant A14C/E45C and A14C/E45C/G89V CA proteins were expressed in Escherichia coli and purified under reducing conditions by anion-exchange chromatography, to > 98% purity ( ). .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ).

Molecular Weight:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: A decrease in SUMO-2/3 conjugation of high molecular weight proteins was evident in the cortex of transgenic mice, and although changes in the modification of multiple SUMO-1 and SUMO-2/3 conjugates were apparent in all brain regions, no other significant changes in conjugation or in levels of SUMO conjugation machinery were observed between transgenic and control mice. .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich).

Mouse Assay:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: Hippocampal, cortical and cerebellar regions were prepared from 9-month male Tg2576 mice and wild-type age-matched controls processed in parallel. .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich).

Transgenic Assay:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: A decrease in SUMO-2/3 conjugation of high molecular weight proteins was evident in the cortex of transgenic mice, and although changes in the modification of multiple SUMO-1 and SUMO-2/3 conjugates were apparent in all brain regions, no other significant changes in conjugation or in levels of SUMO conjugation machinery were observed between transgenic and control mice. .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich).

Lysis:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: .. For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich). .. For the tissue homogenisation protocol, equivalent amounts of tissue were lysed in equivalent buffer volumes, samples were sonicated for 10 s at 4 °C and total protein concentrations were determined as previously reported .

Article Title: IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis
Article Snippet: .. The apical lobe of each lung was resected and immediately homogenized in NP40 lysis buffer (50 mM Tris [pH 7.4], 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3 VO4 ,1% NP40,0.02% NaN3 ) containing a cocktail of mammalian protease inhibitors (Roche). .. For ELISA determination of lung IL12p40 concentration, we used the BD OptEIA Mouse IL-12 (p40) ELISA Set system per the manufacturer's instructions (BD Biosciences).

Purification:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: Recombinant A14C/E45C and A14C/E45C/G89V CA proteins were expressed in Escherichia coli and purified under reducing conditions by anion-exchange chromatography, to > 98% purity ( ). .. To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ).

Chromatin Immunoprecipitation:

Article Title: DNA Methylation Has a Local Effect on Transcription and Histone Acetylation
Article Snippet: Paragraph title: Chromatin immunoprecipitation. ... Approximately 3 × 106 fixed cells were resuspended in 1 ml of radioimmunoprecipitation buffer (10 mM sodium phosphate [pH 7.2], 2 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.2 mM Na3 VO4 , 1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate) that contained mammalian protease inhibitors (Roche).

Article Title: DNA Methylation Dictates Histone H3K4 Methylation ▿
Article Snippet: Paragraph title: Chromatin immunoprecipitation. ... Approximately 5 × 106 fixed cells were resuspended in 1 ml radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) that contained mammalian protease inhibitors (Roche).

SDS Page:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich). .. Equal amounts of protein loaded at 10–50 μg protein/lane were resolved by SDS-PAGE (8–15%) and immunoblotting was performed as formerly described .

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: To check GST-protein expression, a portion of GST-protein-conjugated beads was denatured in 4× LDS (Life Technologies, NP0007) at 70°C for 15 min and subjected to SDS-PAGE followed by Coomassie Blue staining (Invitrogen, LC6060). .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis.

Real-time Polymerase Chain Reaction:

Article Title: DNA Methylation Has a Local Effect on Transcription and Histone Acetylation
Article Snippet: Approximately 3 × 106 fixed cells were resuspended in 1 ml of radioimmunoprecipitation buffer (10 mM sodium phosphate [pH 7.2], 2 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.2 mM Na3 VO4 , 1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate) that contained mammalian protease inhibitors (Roche). .. Aliquots of soluble chromatin, the total chromatin fraction (TCF), were reserved for quantitative PCR (Q-PCR) analysis and for monitoring of the DNA fragment size.

Binding Assay:

Article Title: Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits
Article Snippet: Paragraph title: Assay of TRIMCyp binding to CA tubes. ... To prepare cell extracts containing TRIMCyp, cells were collected from eight confluent 10-cm dishes of cultured 293T cells stably expressing myc-tagged TRIMCyp, resuspended in 2.5 ml of buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol [DTT], mammalian protease inhibitor [Roche]), and lysed by 10 passes with a ball-bearing homogenizer (18-μm gap), as previously described ( ).

Radio Immunoprecipitation:

Article Title: DNA Methylation Dictates Histone H3K4 Methylation ▿
Article Snippet: .. Approximately 5 × 106 fixed cells were resuspended in 1 ml radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) that contained mammalian protease inhibitors (Roche). .. The cell suspension was sonicated and centrifuged as described previously ( ).

Homogenization:

Article Title: Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model
Article Snippet: For each of the regions, both hemispheres were pooled together in ice-cold lysis buffer containing: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% mammalian protease inhibitor (Roche) and 20 mM NEM (Sigma–Aldrich). .. For the tissue homogenisation protocol, equivalent amounts of tissue were lysed in equivalent buffer volumes, samples were sonicated for 10 s at 4 °C and total protein concentrations were determined as previously reported .

Immunoprecipitation:

Article Title: DNA Methylation Dictates Histone H3K4 Methylation ▿
Article Snippet: Approximately 5 × 106 fixed cells were resuspended in 1 ml radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) that contained mammalian protease inhibitors (Roche). .. One 120-μl aliquot of soluble chromatin was used for each immunoprecipitation, and one aliquot was reserved as the total chromatin fraction (TCF) without immunoprecipitation steps for Q-PCR analysis.

Staining:

Article Title: Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche
Article Snippet: To check GST-protein expression, a portion of GST-protein-conjugated beads was denatured in 4× LDS (Life Technologies, NP0007) at 70°C for 15 min and subjected to SDS-PAGE followed by Coomassie Blue staining (Invitrogen, LC6060). .. Beads were washed with PBS containing 1% Triton X-100 and mammalian protease inhibitors (Roche, 11 697 498 001), denatured in 4× LDS at 70°C for 15 min, and subjected to anti-Flag western blot analysis.

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  • 82
    Roche cellytic m mammalian cell lysis extraction reagent
    Overexpression of SMYD3 enhances the AKT pathway A, C.  293T cells (A) and HeLa cells (C) were transfected with Mock vector or SMYD3 expression vector (pcDNA3.1-SMYD3) together with wild-type FLAG-AKT1 expression vector (FLAG-AKT1-WT). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308), anti-phospho mTOR (Ser 2448), anti-mTOR, anti-SMYD3 and anti-FLAG (internal control) antibodies.  B, D.  X-ray films examined in (A) and (C) were scanned with GS-800™ calibrated densitometer (Bio-Rad), and the intensity of monomethylated AKT1 (K14), phospho AKT (Thr 308) and phospho mTOR (Ser 2448) in 293T cells (B) and HeLa cells (D) was normalized by each total protein level.
    Cellytic M Mammalian Cell Lysis Extraction Reagent, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cellytic m mammalian cell lysis extraction reagent/product/Roche
    Average 82 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cellytic m mammalian cell lysis extraction reagent - by Bioz Stars, 2020-03
    82/100 stars
      Buy from Supplier

    79
    Roche mammalian gst pull down
    vMAP Interaction with VDAC1 Inhibits Cytochrome c Release (A) vMAP contains two VDAC1-interacting regions (dark grey boxes). The left panel shows the schematic diagram of vMAP interaction with VDAC1. (Right panel) <t>GST</t> fusions containing various vMAP sequences were expressed and purified from E. coli . <t>293T</t> cells were lysed in CHAPS buffer and subjected to in vitro GST pull-down, followed by immunoblotting with anti-VDAC antibody. The bottom panel shows the Coomassie blue staining of GST fusion proteins. Lanes 1–4 correspond to the GST fusions shown by the left diagram. (B) The LLxL repeat sequences of vMAP are required for VDAC1 interaction. (Top box) The boxed sequences are the two LLxL repeats of vMAP. GST or GST fusion proteins were used to bind to VDAC1 from 293T cell lysates in CHAPS buffer as described in (A). Protein precipitates were analyzed by immunoblotting with anti-VDAC antibody (top blot) and Coomassie blue staining of GST fusion proteins (bottom blot). (C) vMAP interacts with VDAC1 in stable cell lines. NIH3T3 cells stably expressing vector, vMAP, or its mutants were used for immunoprecipitation with anti-vMAP serum or pre-immune (Pre) serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels). (D) vMAP interacts with VDAC1 in γHV-68-infected cells. WCLs of mock (M)- or γHV-68 (γ)-infected (MOI = 1) cells were precipitated with anti-vMAP or preimmune serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels). (E) vMAP inhibits cytochrome c release upon ST treatment. NIH3T3/puro, NIH3T3/vMAP, or NIH3T3/vMAP L/A cells were treated with ST (1 μM) for 4 h and WCLs were sequentially centrifuged to obtain cytosolic (Cyto) and mitochondrion-enriched heavy membrane (HM) fractions. Polypeptides (20 μg) from each fraction were analyzed by immunoblotting with antibodies to cytochrome c (Cyt C), COX4, and actin.
    Mammalian Gst Pull Down, supplied by Roche, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mammalian gst pull down/product/Roche
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mammalian gst pull down - by Bioz Stars, 2020-03
    79/100 stars
      Buy from Supplier

    84
    Roche mclb np 40 buffer
    Association of SLC38A9 with Rag GTPases is amino acid and nucleotide dependent. (A) 293T-REx cells inducibly expressing HA-tagged Rag GTPase proteins were transfected with GFP-SLC38A9 full length, GFP-SLC38A9 aa 1 to 119, or GFP alone. Cells were either grown for 50 min in full RPMI growth medium (fed) or were amino acid (aa) starved and subsequently lysed with <t>MCLB</t> <t>NP-40</t> for GFP immunoprecipitation. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. (B) 293T cells were transfected with nontargeting control (sicon) or SLC38A9 siRNAs and myc-tagged LAMTOR2, as well as wild-type (RRAGB wild-type [RB wt], RRAGC wild-type [RC wt], RRAGA wild type [RA wt]) or mutant (RRAGB Q99L [RB QL], RRAGB T75L [RB TL], RRAGC Q120L [RC QL], RRAGC S54L [RC SL]) HA-tagged Rag GTPase proteins, as indicated. Cells were lysed in HEPES buffer, subjected to HA-immunoprecipitation, and analyzed as described above. (C) 293T-REx cells inducibly expressing HA-tagged RRAGC, LAMTOR1, or Raptor (marked with red edging) were transfected with control (sicon) or SLC38A9 siRNA as indicated, followed by lysis in MCLB CHAPS buffer and immunoprecipitation with HA beads. Eluates were analyzed by MS. The number of peptides is shown from 0 to 100 peptides per protein. See Table S1D in the supplemental material for complete proteomic data. (D) Cells like those described for panel C were lysed in HEPES buffer followed by immunoprecipitation, SDS-PAGE, and immunoblotting. (E) Raptor overexpressing 293T-REx cells were transfected as described for panel C, lysed in MCLB CHAPS buffer, and analyzed as described for panel D.
    Mclb Np 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Roche mammalian disaggregase
    Mammalian amyloid <t>disaggregase</t> and proteolysis activities are sensitive to low and high pH pretreatment. Dialysis of HEK 293 PNS (1 mg/mL total protein) for 16 h at 4 °C into buffers at low pH (and to a lesser extent high pH) decreases its ability
    Mammalian Disaggregase, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mammalian disaggregase/product/Roche
    Average 78 stars, based on 1 article reviews
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    Image Search Results


    Overexpression of SMYD3 enhances the AKT pathway A, C.  293T cells (A) and HeLa cells (C) were transfected with Mock vector or SMYD3 expression vector (pcDNA3.1-SMYD3) together with wild-type FLAG-AKT1 expression vector (FLAG-AKT1-WT). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308), anti-phospho mTOR (Ser 2448), anti-mTOR, anti-SMYD3 and anti-FLAG (internal control) antibodies.  B, D.  X-ray films examined in (A) and (C) were scanned with GS-800™ calibrated densitometer (Bio-Rad), and the intensity of monomethylated AKT1 (K14), phospho AKT (Thr 308) and phospho mTOR (Ser 2448) in 293T cells (B) and HeLa cells (D) was normalized by each total protein level.

    Journal: Oncotarget

    Article Title: SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    doi: 10.18632/oncotarget.11898

    Figure Lengend Snippet: Overexpression of SMYD3 enhances the AKT pathway A, C. 293T cells (A) and HeLa cells (C) were transfected with Mock vector or SMYD3 expression vector (pcDNA3.1-SMYD3) together with wild-type FLAG-AKT1 expression vector (FLAG-AKT1-WT). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308), anti-phospho mTOR (Ser 2448), anti-mTOR, anti-SMYD3 and anti-FLAG (internal control) antibodies. B, D. X-ray films examined in (A) and (C) were scanned with GS-800™ calibrated densitometer (Bio-Rad), and the intensity of monomethylated AKT1 (K14), phospho AKT (Thr 308) and phospho mTOR (Ser 2448) in 293T cells (B) and HeLa cells (D) was normalized by each total protein level.

    Article Snippet: After 72 hours of incubation, cells were treated with 100 ng/ml of EGFR (SW480) and 100 ng/ml neuregulin 1 (NRG1, MDA-MB-231) for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent with a complete protease inhibitor cocktail (Roche Applied Science) and a phosphatase inhibitor cocktail (Roche Applied Science).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Incubation, Lysis, Protease Inhibitor

    SMYD3-mediated lysine 14 methylation is critical for AKT1 activation A.  293T cells were transfected with FLAG-tagged wild-type AKT1 (AKT1-WT) or mutant-type AKT1 (AKT1-K14A, AKT1-K30A, AKT1-K39A, AKT1-K14A/K30A, AKT1-K14A/K39A, AKT1-K30A/K39A or AKT1-K14A/K30A/K39A) in the presence of a wild-type SMYD3 expression vector (pcDNA-SMYD3.1-WT). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail, followed by immunoprecipitation using anti-FLAG ®  M2 affinity gel. Immunoprecipitates were immunoblotted with anti-phospho AKT (Thr 308) and anti-FLAG (internal control) antibodies.  B.  The three-dimensional coordinate data for AKT1 were derived from the Protein Data Bank (entry code, 4EJN) [  59 ]. PH domain, activation loop, kinase domain and ATP binding site are described.  C.  Lysine 14 methylation can conformationally affect threonine 308. Lys 14 is hydrogen-bounding to Glu 17. The distance between the side-chain amino nitrogen atom (Nζ) of Lys 14 and the side-chain carboxyl oxygen atom (Oε2) of Glu 17 is 3.17 Å. Glu 17 is in van der Waals contact with the activation loop (Thr 291 – Glu 314), on which Thr 308 is located. The distance between their closest atoms (the backbone carboxyl oxygen atom of Glu 17 and the side-chain β carbon atom of Phe 309) is 3.35 Å. A part of the activation loop (Asp 302 – Met 306) is missing in the crystal structure. The drawing was created using  Molecular Operating Environment  (MOE), 2014.09 (Chemical Computing Group Inc., Canada).  D.  The distance between Lys 14, Lys 30 or Lys 39 in the PH domain and activation loop is described.

    Journal: Oncotarget

    Article Title: SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    doi: 10.18632/oncotarget.11898

    Figure Lengend Snippet: SMYD3-mediated lysine 14 methylation is critical for AKT1 activation A. 293T cells were transfected with FLAG-tagged wild-type AKT1 (AKT1-WT) or mutant-type AKT1 (AKT1-K14A, AKT1-K30A, AKT1-K39A, AKT1-K14A/K30A, AKT1-K14A/K39A, AKT1-K30A/K39A or AKT1-K14A/K30A/K39A) in the presence of a wild-type SMYD3 expression vector (pcDNA-SMYD3.1-WT). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail, followed by immunoprecipitation using anti-FLAG ® M2 affinity gel. Immunoprecipitates were immunoblotted with anti-phospho AKT (Thr 308) and anti-FLAG (internal control) antibodies. B. The three-dimensional coordinate data for AKT1 were derived from the Protein Data Bank (entry code, 4EJN) [ 59 ]. PH domain, activation loop, kinase domain and ATP binding site are described. C. Lysine 14 methylation can conformationally affect threonine 308. Lys 14 is hydrogen-bounding to Glu 17. The distance between the side-chain amino nitrogen atom (Nζ) of Lys 14 and the side-chain carboxyl oxygen atom (Oε2) of Glu 17 is 3.17 Å. Glu 17 is in van der Waals contact with the activation loop (Thr 291 – Glu 314), on which Thr 308 is located. The distance between their closest atoms (the backbone carboxyl oxygen atom of Glu 17 and the side-chain β carbon atom of Phe 309) is 3.35 Å. A part of the activation loop (Asp 302 – Met 306) is missing in the crystal structure. The drawing was created using Molecular Operating Environment (MOE), 2014.09 (Chemical Computing Group Inc., Canada). D. The distance between Lys 14, Lys 30 or Lys 39 in the PH domain and activation loop is described.

    Article Snippet: After 72 hours of incubation, cells were treated with 100 ng/ml of EGFR (SW480) and 100 ng/ml neuregulin 1 (NRG1, MDA-MB-231) for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent with a complete protease inhibitor cocktail (Roche Applied Science) and a phosphatase inhibitor cocktail (Roche Applied Science).

    Techniques: Methylation, Activation Assay, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Lysis, Protease Inhibitor, Immunoprecipitation, Derivative Assay, Binding Assay

    Validation of methylation on AKT1 at lysine 14 by specific antibody A.  The enzyme-linked immunosorbent assay of the anti-monomethyl AKT1 (Lys 14) antibody. A rabbit was immunized with synthetic peptides, including Lys 14 monomethylation, and the affinity purification was carried out against the methylated synthetic peptides. The original serum (S) and the flow through (FT) show equal reactivity against the carrier protein. The purified antibody (PA) shows a stronger reactivity against the methylated peptide than the serum (S).  B.  Testing of specific antibody (SA) and non-specific antibody (NS) against the unmodified peptide by enzyme-linked immunosorbent assay.  C.  Validation of the anti-K14 monomethylated AKT1 antibody. Recombinant AKT1 protein or BSA and S-adenosyl-L-methionine were incubated in the presence or absence of recombinant SMYD3, and the reaction products were analyzed by SDS-PAGE, followed by western blot analysis using the anti-K14 monomethylated AKT1 antibody. The nitrocellulose membrane was stained with MemCode™ Reversible Stain Kit after western blot analysis.  D.  293T cells were co-transfected with Mock, FLAG-AKT1-WT, FLAG-AKT1-K14A or FLAG-AKT1-K14R together with a wild-type SMYD3 expression vector (pcDNA3.1-SMYD3-WT) or an enzyme-inactive SMYD3 expression (pcDNA3.1-SMYD3-ΔEEL). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308) and anti-FLAG antibodies after immunoprecipitating with anti-FLAG ®  M2 affinity gel.

    Journal: Oncotarget

    Article Title: SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    doi: 10.18632/oncotarget.11898

    Figure Lengend Snippet: Validation of methylation on AKT1 at lysine 14 by specific antibody A. The enzyme-linked immunosorbent assay of the anti-monomethyl AKT1 (Lys 14) antibody. A rabbit was immunized with synthetic peptides, including Lys 14 monomethylation, and the affinity purification was carried out against the methylated synthetic peptides. The original serum (S) and the flow through (FT) show equal reactivity against the carrier protein. The purified antibody (PA) shows a stronger reactivity against the methylated peptide than the serum (S). B. Testing of specific antibody (SA) and non-specific antibody (NS) against the unmodified peptide by enzyme-linked immunosorbent assay. C. Validation of the anti-K14 monomethylated AKT1 antibody. Recombinant AKT1 protein or BSA and S-adenosyl-L-methionine were incubated in the presence or absence of recombinant SMYD3, and the reaction products were analyzed by SDS-PAGE, followed by western blot analysis using the anti-K14 monomethylated AKT1 antibody. The nitrocellulose membrane was stained with MemCode™ Reversible Stain Kit after western blot analysis. D. 293T cells were co-transfected with Mock, FLAG-AKT1-WT, FLAG-AKT1-K14A or FLAG-AKT1-K14R together with a wild-type SMYD3 expression vector (pcDNA3.1-SMYD3-WT) or an enzyme-inactive SMYD3 expression (pcDNA3.1-SMYD3-ΔEEL). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308) and anti-FLAG antibodies after immunoprecipitating with anti-FLAG ® M2 affinity gel.

    Article Snippet: After 72 hours of incubation, cells were treated with 100 ng/ml of EGFR (SW480) and 100 ng/ml neuregulin 1 (NRG1, MDA-MB-231) for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent with a complete protease inhibitor cocktail (Roche Applied Science) and a phosphatase inhibitor cocktail (Roche Applied Science).

    Techniques: Methylation, Enzyme-linked Immunosorbent Assay, Affinity Purification, Flow Cytometry, Purification, Peptide ELISA, Recombinant, Incubation, SDS Page, Western Blot, Staining, Transfection, Expressing, Plasmid Preparation, Lysis, Protease Inhibitor

    Knockdown and enzyme inhibitory of SMYD3 attenuate AKT1 activity A, C.  Effects of SMYD3 knockdown on the AKT pathway in SW480 cells (A) and MDA-MB-231 cells (C). Cells were transfected with one control siRNA (siEGFP) or two SMYD3 siRNAs (#1 and #2), and after 72 hours of incubation, cells were treated with 100 ng/ml of EGF (SW480) or 100 ng/ml of NRG1 (MDA-MB-231). Then cells were lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail, followed by SDS-PAGE. Western blot analysis was performed using anti-SMYD3, anti-K14 monomethylated AKT1, anti-phospho AKT1 (Thr 308), anti-AKT1, anti-phospho mTOR (Ser 2448), anti-mTOR and anti-α-Tubulin (internal control) antibodies.  B, D.  X-ray films were scanned with GS-800™ calibrated densitometer (Bio-Rad). The intensity of phosphorylated AKT (Thr 308) and phosphorylated mTOR (Ser 2448) levels in SW480 cells (B) and MDA-MB-231 cells (D) was normalized by each total protein level.  E, G.  Effects of BCI-121 treatment on the AKT1 activity. SW480 cells (E) and MDA-MB-231 cells (G) were treated with 0, 10 or 50 μM of BCI-121. After 72 hours of incubation, cells were treated with 100 ng/ml of EGF (SW480) or 100 ng/ml of NRG1 (MDA-MB-231), and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. Cell extracts were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308), anti-AKT and anti-α-Tubulin (internal control) antibodies.  F, H.  X-ray films were scanned with GS-800™ calibrated densitometer (Bio-Rad). The intensity of methylated AKT1 (Lys 14) and phosphorylated AKT1 (Thr 308) levels in SW480 cells (F) and MDA-MB-231 cells (H) was normalized by each total protein level.

    Journal: Oncotarget

    Article Title: SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    doi: 10.18632/oncotarget.11898

    Figure Lengend Snippet: Knockdown and enzyme inhibitory of SMYD3 attenuate AKT1 activity A, C. Effects of SMYD3 knockdown on the AKT pathway in SW480 cells (A) and MDA-MB-231 cells (C). Cells were transfected with one control siRNA (siEGFP) or two SMYD3 siRNAs (#1 and #2), and after 72 hours of incubation, cells were treated with 100 ng/ml of EGF (SW480) or 100 ng/ml of NRG1 (MDA-MB-231). Then cells were lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail, followed by SDS-PAGE. Western blot analysis was performed using anti-SMYD3, anti-K14 monomethylated AKT1, anti-phospho AKT1 (Thr 308), anti-AKT1, anti-phospho mTOR (Ser 2448), anti-mTOR and anti-α-Tubulin (internal control) antibodies. B, D. X-ray films were scanned with GS-800™ calibrated densitometer (Bio-Rad). The intensity of phosphorylated AKT (Thr 308) and phosphorylated mTOR (Ser 2448) levels in SW480 cells (B) and MDA-MB-231 cells (D) was normalized by each total protein level. E, G. Effects of BCI-121 treatment on the AKT1 activity. SW480 cells (E) and MDA-MB-231 cells (G) were treated with 0, 10 or 50 μM of BCI-121. After 72 hours of incubation, cells were treated with 100 ng/ml of EGF (SW480) or 100 ng/ml of NRG1 (MDA-MB-231), and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. Cell extracts were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308), anti-AKT and anti-α-Tubulin (internal control) antibodies. F, H. X-ray films were scanned with GS-800™ calibrated densitometer (Bio-Rad). The intensity of methylated AKT1 (Lys 14) and phosphorylated AKT1 (Thr 308) levels in SW480 cells (F) and MDA-MB-231 cells (H) was normalized by each total protein level.

    Article Snippet: After 72 hours of incubation, cells were treated with 100 ng/ml of EGFR (SW480) and 100 ng/ml neuregulin 1 (NRG1, MDA-MB-231) for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent with a complete protease inhibitor cocktail (Roche Applied Science) and a phosphatase inhibitor cocktail (Roche Applied Science).

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Incubation, Lysis, Protease Inhibitor, SDS Page, Western Blot, Methylation

    vMAP Interaction with VDAC1 Inhibits Cytochrome c Release (A) vMAP contains two VDAC1-interacting regions (dark grey boxes). The left panel shows the schematic diagram of vMAP interaction with VDAC1. (Right panel) GST fusions containing various vMAP sequences were expressed and purified from E. coli . 293T cells were lysed in CHAPS buffer and subjected to in vitro GST pull-down, followed by immunoblotting with anti-VDAC antibody. The bottom panel shows the Coomassie blue staining of GST fusion proteins. Lanes 1–4 correspond to the GST fusions shown by the left diagram. (B) The LLxL repeat sequences of vMAP are required for VDAC1 interaction. (Top box) The boxed sequences are the two LLxL repeats of vMAP. GST or GST fusion proteins were used to bind to VDAC1 from 293T cell lysates in CHAPS buffer as described in (A). Protein precipitates were analyzed by immunoblotting with anti-VDAC antibody (top blot) and Coomassie blue staining of GST fusion proteins (bottom blot). (C) vMAP interacts with VDAC1 in stable cell lines. NIH3T3 cells stably expressing vector, vMAP, or its mutants were used for immunoprecipitation with anti-vMAP serum or pre-immune (Pre) serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels). (D) vMAP interacts with VDAC1 in γHV-68-infected cells. WCLs of mock (M)- or γHV-68 (γ)-infected (MOI = 1) cells were precipitated with anti-vMAP or preimmune serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels). (E) vMAP inhibits cytochrome c release upon ST treatment. NIH3T3/puro, NIH3T3/vMAP, or NIH3T3/vMAP L/A cells were treated with ST (1 μM) for 4 h and WCLs were sequentially centrifuged to obtain cytosolic (Cyto) and mitochondrion-enriched heavy membrane (HM) fractions. Polypeptides (20 μg) from each fraction were analyzed by immunoblotting with antibodies to cytochrome c (Cyt C), COX4, and actin.

    Journal: PLoS Pathogens

    Article Title: A Novel Inhibitory Mechanism of Mitochondrion-Dependent Apoptosis by a Herpesviral Protein

    doi: 10.1371/journal.ppat.0030174

    Figure Lengend Snippet: vMAP Interaction with VDAC1 Inhibits Cytochrome c Release (A) vMAP contains two VDAC1-interacting regions (dark grey boxes). The left panel shows the schematic diagram of vMAP interaction with VDAC1. (Right panel) GST fusions containing various vMAP sequences were expressed and purified from E. coli . 293T cells were lysed in CHAPS buffer and subjected to in vitro GST pull-down, followed by immunoblotting with anti-VDAC antibody. The bottom panel shows the Coomassie blue staining of GST fusion proteins. Lanes 1–4 correspond to the GST fusions shown by the left diagram. (B) The LLxL repeat sequences of vMAP are required for VDAC1 interaction. (Top box) The boxed sequences are the two LLxL repeats of vMAP. GST or GST fusion proteins were used to bind to VDAC1 from 293T cell lysates in CHAPS buffer as described in (A). Protein precipitates were analyzed by immunoblotting with anti-VDAC antibody (top blot) and Coomassie blue staining of GST fusion proteins (bottom blot). (C) vMAP interacts with VDAC1 in stable cell lines. NIH3T3 cells stably expressing vector, vMAP, or its mutants were used for immunoprecipitation with anti-vMAP serum or pre-immune (Pre) serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels). (D) vMAP interacts with VDAC1 in γHV-68-infected cells. WCLs of mock (M)- or γHV-68 (γ)-infected (MOI = 1) cells were precipitated with anti-vMAP or preimmune serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels). (E) vMAP inhibits cytochrome c release upon ST treatment. NIH3T3/puro, NIH3T3/vMAP, or NIH3T3/vMAP L/A cells were treated with ST (1 μM) for 4 h and WCLs were sequentially centrifuged to obtain cytosolic (Cyto) and mitochondrion-enriched heavy membrane (HM) fractions. Polypeptides (20 μg) from each fraction were analyzed by immunoblotting with antibodies to cytochrome c (Cyt C), COX4, and actin.

    Article Snippet: For mammalian GST pull-down, 293T cells expressing GST fusion proteins and Bcl-2 family proteins were harvested and lysed with 1% CHAPS buffer (50 mM HEPES [pH 7.4]; 100 mM NaCl; 10 mM Tris; 1 mM EDTA; 1% CHAPS) supplemented with protease inhibitor cocktail (Roche).

    Techniques: Purification, In Vitro, Staining, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Infection

    vMAP Interacts with Bcl-2/Bcl-x L (A) vMAP interacts with Bcl-2/Bcl-x L in γHV-68-infected cells. At 16 h post-infection, γHV-68-infected (MOI = 1) or mock-infected NIH3T3 cells were harvested and post-centrifuge supernatants were subjected to immunoprecipitation (IP) with rabbit antibodies to Bcl-2 or Bcl-x L , followed by immunoblotting with anti-vMAP serum. A normal rabbit serum was included as a negative control. Protein precipitates were analyzed by immunoblotting with antibodies to Bcl-2 and Bcl-x L (middle panel). Whole cell lysates (WCL) of mock-infected or γHV-68-infected NIH3T3 cells were immunoblotted with anti-vMAP serum (bottom panel). (B) The N-terminal sequence of vMAP interacts with Bcl-2/Bcl-x L but not with Bak and Bid. At 48 h post-transfection with a plasmid expressing HA-Bcl-2, HA-Bcl-x L , HA-Bak, or HA-Bid, and a plasmid expressing GST (lane 2) or vMAP(1–50)-GST (lane 3), cell lysates of 293T were used for GST pull-down (PD) assay, followed by immunoblotting with anti-HA (top panel of each set). WCLs were analyzed by immunoblotting with antibodies to HA epitope (Bcl-2 family proteins, middle panels) and GST (bottom panels) for GST or vMAP(1–50)-GST expression. Lane 1 is 2% input of lysates of cells expressing GST and Bcl-2 family proteins. For all four sets of PDs (top panel), lane 1 shows the equivalent amount of Bcl-2 family proteins compared to those shown in the middle panel. (C) The first 20 amino acids of vMAP are essential for its interaction with Bcl-2/Bcl-x L . 293T cells were transfected with a plasmid expressing HA-Bcl-2, HA-Bcl-x L together with a plasmid expressing GST (lane 1), vMAP-GST (lane 2), or vMAPΔ20-GST (lane 3). Mammalian GST pull-down was carried out as described in (B). The precipitates were analyzed by immunoblotting with anti-HA to detect Bcl-2 and Bcl-x L (top two panels). WCLs were analyzed by immunoblotting with anti-HA and anti-GST antibodies to demonstrate the expression of Bcl-2/Bcl-x L and GST fusion, respectively. (D) The Bcl-2 BH2 domain is not involved in vMAP binding. At 48 h post-transfection with a plasmid expressing HA-Bcl-2, Bcl-2 G 145A , or Bcl-2 W 188A together with a plasmid expressing GST (lane 2) or vMAP(1–50)-GST (lane 3), 293T cell lysates were used for GST pull-down, followed by immunoblotting with Bcl-2 antibody (top panel of each set). WCLs were analyzed by immunoblotting with antibodies to Bcl-2 (middle panel) and GST (bottom panel). Lane 1 indicates 2% input of lysates of cells expressing vMAP-GST and Bcl-2/Bcl-x L .

    Journal: PLoS Pathogens

    Article Title: A Novel Inhibitory Mechanism of Mitochondrion-Dependent Apoptosis by a Herpesviral Protein

    doi: 10.1371/journal.ppat.0030174

    Figure Lengend Snippet: vMAP Interacts with Bcl-2/Bcl-x L (A) vMAP interacts with Bcl-2/Bcl-x L in γHV-68-infected cells. At 16 h post-infection, γHV-68-infected (MOI = 1) or mock-infected NIH3T3 cells were harvested and post-centrifuge supernatants were subjected to immunoprecipitation (IP) with rabbit antibodies to Bcl-2 or Bcl-x L , followed by immunoblotting with anti-vMAP serum. A normal rabbit serum was included as a negative control. Protein precipitates were analyzed by immunoblotting with antibodies to Bcl-2 and Bcl-x L (middle panel). Whole cell lysates (WCL) of mock-infected or γHV-68-infected NIH3T3 cells were immunoblotted with anti-vMAP serum (bottom panel). (B) The N-terminal sequence of vMAP interacts with Bcl-2/Bcl-x L but not with Bak and Bid. At 48 h post-transfection with a plasmid expressing HA-Bcl-2, HA-Bcl-x L , HA-Bak, or HA-Bid, and a plasmid expressing GST (lane 2) or vMAP(1–50)-GST (lane 3), cell lysates of 293T were used for GST pull-down (PD) assay, followed by immunoblotting with anti-HA (top panel of each set). WCLs were analyzed by immunoblotting with antibodies to HA epitope (Bcl-2 family proteins, middle panels) and GST (bottom panels) for GST or vMAP(1–50)-GST expression. Lane 1 is 2% input of lysates of cells expressing GST and Bcl-2 family proteins. For all four sets of PDs (top panel), lane 1 shows the equivalent amount of Bcl-2 family proteins compared to those shown in the middle panel. (C) The first 20 amino acids of vMAP are essential for its interaction with Bcl-2/Bcl-x L . 293T cells were transfected with a plasmid expressing HA-Bcl-2, HA-Bcl-x L together with a plasmid expressing GST (lane 1), vMAP-GST (lane 2), or vMAPΔ20-GST (lane 3). Mammalian GST pull-down was carried out as described in (B). The precipitates were analyzed by immunoblotting with anti-HA to detect Bcl-2 and Bcl-x L (top two panels). WCLs were analyzed by immunoblotting with anti-HA and anti-GST antibodies to demonstrate the expression of Bcl-2/Bcl-x L and GST fusion, respectively. (D) The Bcl-2 BH2 domain is not involved in vMAP binding. At 48 h post-transfection with a plasmid expressing HA-Bcl-2, Bcl-2 G 145A , or Bcl-2 W 188A together with a plasmid expressing GST (lane 2) or vMAP(1–50)-GST (lane 3), 293T cell lysates were used for GST pull-down, followed by immunoblotting with Bcl-2 antibody (top panel of each set). WCLs were analyzed by immunoblotting with antibodies to Bcl-2 (middle panel) and GST (bottom panel). Lane 1 indicates 2% input of lysates of cells expressing vMAP-GST and Bcl-2/Bcl-x L .

    Article Snippet: For mammalian GST pull-down, 293T cells expressing GST fusion proteins and Bcl-2 family proteins were harvested and lysed with 1% CHAPS buffer (50 mM HEPES [pH 7.4]; 100 mM NaCl; 10 mM Tris; 1 mM EDTA; 1% CHAPS) supplemented with protease inhibitor cocktail (Roche).

    Techniques: Infection, Immunoprecipitation, Negative Control, Sequencing, Transfection, Plasmid Preparation, Expressing, Binding Assay

    Association of SLC38A9 with Rag GTPases is amino acid and nucleotide dependent. (A) 293T-REx cells inducibly expressing HA-tagged Rag GTPase proteins were transfected with GFP-SLC38A9 full length, GFP-SLC38A9 aa 1 to 119, or GFP alone. Cells were either grown for 50 min in full RPMI growth medium (fed) or were amino acid (aa) starved and subsequently lysed with MCLB NP-40 for GFP immunoprecipitation. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. (B) 293T cells were transfected with nontargeting control (sicon) or SLC38A9 siRNAs and myc-tagged LAMTOR2, as well as wild-type (RRAGB wild-type [RB wt], RRAGC wild-type [RC wt], RRAGA wild type [RA wt]) or mutant (RRAGB Q99L [RB QL], RRAGB T75L [RB TL], RRAGC Q120L [RC QL], RRAGC S54L [RC SL]) HA-tagged Rag GTPase proteins, as indicated. Cells were lysed in HEPES buffer, subjected to HA-immunoprecipitation, and analyzed as described above. (C) 293T-REx cells inducibly expressing HA-tagged RRAGC, LAMTOR1, or Raptor (marked with red edging) were transfected with control (sicon) or SLC38A9 siRNA as indicated, followed by lysis in MCLB CHAPS buffer and immunoprecipitation with HA beads. Eluates were analyzed by MS. The number of peptides is shown from 0 to 100 peptides per protein. See Table S1D in the supplemental material for complete proteomic data. (D) Cells like those described for panel C were lysed in HEPES buffer followed by immunoprecipitation, SDS-PAGE, and immunoblotting. (E) Raptor overexpressing 293T-REx cells were transfected as described for panel C, lysed in MCLB CHAPS buffer, and analyzed as described for panel D.

    Journal: Molecular and Cellular Biology

    Article Title: Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9

    doi: 10.1128/MCB.00125-15

    Figure Lengend Snippet: Association of SLC38A9 with Rag GTPases is amino acid and nucleotide dependent. (A) 293T-REx cells inducibly expressing HA-tagged Rag GTPase proteins were transfected with GFP-SLC38A9 full length, GFP-SLC38A9 aa 1 to 119, or GFP alone. Cells were either grown for 50 min in full RPMI growth medium (fed) or were amino acid (aa) starved and subsequently lysed with MCLB NP-40 for GFP immunoprecipitation. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. (B) 293T cells were transfected with nontargeting control (sicon) or SLC38A9 siRNAs and myc-tagged LAMTOR2, as well as wild-type (RRAGB wild-type [RB wt], RRAGC wild-type [RC wt], RRAGA wild type [RA wt]) or mutant (RRAGB Q99L [RB QL], RRAGB T75L [RB TL], RRAGC Q120L [RC QL], RRAGC S54L [RC SL]) HA-tagged Rag GTPase proteins, as indicated. Cells were lysed in HEPES buffer, subjected to HA-immunoprecipitation, and analyzed as described above. (C) 293T-REx cells inducibly expressing HA-tagged RRAGC, LAMTOR1, or Raptor (marked with red edging) were transfected with control (sicon) or SLC38A9 siRNA as indicated, followed by lysis in MCLB CHAPS buffer and immunoprecipitation with HA beads. Eluates were analyzed by MS. The number of peptides is shown from 0 to 100 peptides per protein. See Table S1D in the supplemental material for complete proteomic data. (D) Cells like those described for panel C were lysed in HEPES buffer followed by immunoprecipitation, SDS-PAGE, and immunoblotting. (E) Raptor overexpressing 293T-REx cells were transfected as described for panel C, lysed in MCLB CHAPS buffer, and analyzed as described for panel D.

    Article Snippet: Cell pellets were lysed as indicated in ice-cold MCLB NP-40 buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40), MCLB 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.3% CHAPS), or HEPES buffer (1% NP-40, 50 mM HEPES, pH 7.4, 250 mM NaCl, 5 mM EDTA) supplemented with cOmplete EDTA-free protease inhibitor tablets (Roche) and phosphatase inhibitor (PhosSTOP; Roche) and incubated on ice for 30 min.

    Techniques: Expressing, Transfection, Immunoprecipitation, SDS Page, Mutagenesis, Lysis, Mass Spectrometry

    Interaction proteomics of regulatory mTORC1 components. (A to C) Grouped interaction network schemes obtained by IP-MS analysis of 12 baits consisting of the Rag GTPases (A), all Ragulator subunits (B), and TSC (C) and their high-confidence candidate interacting proteins (HCIPs; average APSM of ≥3 and WD N score of ≥1). All interactions are color-coded according to functional group (red, mTORC1; blue, Ragulator; green, Rag proteins; orange, TSC; black, SLC38A9). Line thickness indicates interactions with WD N scores between 1 and 10. Differential lysis with MCLB NP-40 (solid lines) or MCLB CHAPS (dashed lines) buffer is indicated. See Table S1A and B and Fig. S1 in the supplemental material for complete proteomic data.

    Journal: Molecular and Cellular Biology

    Article Title: Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9

    doi: 10.1128/MCB.00125-15

    Figure Lengend Snippet: Interaction proteomics of regulatory mTORC1 components. (A to C) Grouped interaction network schemes obtained by IP-MS analysis of 12 baits consisting of the Rag GTPases (A), all Ragulator subunits (B), and TSC (C) and their high-confidence candidate interacting proteins (HCIPs; average APSM of ≥3 and WD N score of ≥1). All interactions are color-coded according to functional group (red, mTORC1; blue, Ragulator; green, Rag proteins; orange, TSC; black, SLC38A9). Line thickness indicates interactions with WD N scores between 1 and 10. Differential lysis with MCLB NP-40 (solid lines) or MCLB CHAPS (dashed lines) buffer is indicated. See Table S1A and B and Fig. S1 in the supplemental material for complete proteomic data.

    Article Snippet: Cell pellets were lysed as indicated in ice-cold MCLB NP-40 buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40), MCLB 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.3% CHAPS), or HEPES buffer (1% NP-40, 50 mM HEPES, pH 7.4, 250 mM NaCl, 5 mM EDTA) supplemented with cOmplete EDTA-free protease inhibitor tablets (Roche) and phosphatase inhibitor (PhosSTOP; Roche) and incubated on ice for 30 min.

    Techniques: Mass Spectrometry, Functional Assay, Lysis

    Mammalian amyloid disaggregase and proteolysis activities are sensitive to low and high pH pretreatment. Dialysis of HEK 293 PNS (1 mg/mL total protein) for 16 h at 4 °C into buffers at low pH (and to a lesser extent high pH) decreases its ability

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Discovery and characterization of a mammalian amyloid disaggregation activity

    doi: 10.1002/pro.363

    Figure Lengend Snippet: Mammalian amyloid disaggregase and proteolysis activities are sensitive to low and high pH pretreatment. Dialysis of HEK 293 PNS (1 mg/mL total protein) for 16 h at 4 °C into buffers at low pH (and to a lesser extent high pH) decreases its ability

    Article Snippet: Mammalian disaggregase and proteolysis activities can be uncoupled in the presence of Roche Complete EDTA-Free Protease Inhibitor Cocktail, suggesting that mammalian amyloid disaggregation activity is not driven by proteolysis.

    Techniques:

    Mammalian amyloid disaggregase and proteolysis activities are resistant to high-temperature pretreatment. (Error bars indicate standard deviations in three independent disaggregation or proteolysis experiments; * denotes p

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Discovery and characterization of a mammalian amyloid disaggregation activity

    doi: 10.1002/pro.363

    Figure Lengend Snippet: Mammalian amyloid disaggregase and proteolysis activities are resistant to high-temperature pretreatment. (Error bars indicate standard deviations in three independent disaggregation or proteolysis experiments; * denotes p

    Article Snippet: Mammalian disaggregase and proteolysis activities can be uncoupled in the presence of Roche Complete EDTA-Free Protease Inhibitor Cocktail, suggesting that mammalian amyloid disaggregation activity is not driven by proteolysis.

    Techniques:

    PNS-mediated amyloid disaggregase activity is sensitive to proteolytic digestion. Proteinase K (PK) (2 μg/mL) pretreatment of mouse kidney PNS (300 μg/mL total protein) eliminates its ability (at 10 μg/mL total protein) to disaggregate

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Discovery and characterization of a mammalian amyloid disaggregation activity

    doi: 10.1002/pro.363

    Figure Lengend Snippet: PNS-mediated amyloid disaggregase activity is sensitive to proteolytic digestion. Proteinase K (PK) (2 μg/mL) pretreatment of mouse kidney PNS (300 μg/mL total protein) eliminates its ability (at 10 μg/mL total protein) to disaggregate

    Article Snippet: Mammalian disaggregase and proteolysis activities can be uncoupled in the presence of Roche Complete EDTA-Free Protease Inhibitor Cocktail, suggesting that mammalian amyloid disaggregation activity is not driven by proteolysis.

    Techniques: Activity Assay