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Roche complete antiprotease cocktail roche
Complete Antiprotease Cocktail Roche, supplied by Roche, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Related Articles

Clone Assay:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: Virus release was assayed in HeLa cells, which were transfected with proviral clones using Polyjet (SignaGen Laboratories). .. After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG.

Transfection:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: Virus release was assayed in HeLa cells, which were transfected with proviral clones using Polyjet (SignaGen Laboratories). .. After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG.

Centrifugation:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG. .. Cell-associated material was also lysed in lysis buffer, insoluble material removed by centrifugation, and the soluble material immunoprecipitated with anti-HIV-1 IgG.

Labeling:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: .. After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG. .. Cell-associated material was also lysed in lysis buffer, insoluble material removed by centrifugation, and the soluble material immunoprecipitated with anti-HIV-1 IgG.

Immunoprecipitation:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: .. After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG. .. Cell-associated material was also lysed in lysis buffer, insoluble material removed by centrifugation, and the soluble material immunoprecipitated with anti-HIV-1 IgG.

Infection:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: Paragraph title: Virus release and infectivity ... After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG.

Lysis:

Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation
Article Snippet: .. After 3 h labeling, supernatant was filtered and pelleted, then resuspended in lysis buffer (0.5% Triton X100, 300 mM NaCl, 10 mM iodoacetamide, 50 mM Tris pH 7.5, supplemented with 1} Complete Antiprotease Cocktail - Roche) and immunoprecipitated with anti-HIV-1 IgG. .. Cell-associated material was also lysed in lysis buffer, insoluble material removed by centrifugation, and the soluble material immunoprecipitated with anti-HIV-1 IgG.

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  • 99
    Roche edta free protease inhibitor cocktail tablets
    Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in <t>PBS</t> alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M <t>EDTA,</t> were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .
    Edta Free Protease Inhibitor Cocktail Tablets, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche edta free complete protease inhibitor cocktail
    Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by <t>SDS-polyacrylamide</t> gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM <t>EDTA</t> (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.
    Edta Free Complete Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche immunoprecipitation buffer
    The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) <t>Immunoprecipitation</t> of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche tnex buffer
    Evidence for CPAF mediated processing of vimentin in intact cells A) CPAF is not active in 1% SDS buffer. <t>HeLa</t> cell lysates prepared under denaturing conditions in 1% SDS buffer were incubated with the 0.5, 2.5, and 5 μg recombinant CPAF (rCPAF) for 20 minutes at 37 ° C. As a positive control, rCPAF was also incubated with HeLa lysates prepared under non-denaturing conditions in <t>TNEX</t> buffer. Where indicated, 100 μM CPAF inhibitor peptide (CIP) was included as a control. CPAF activity was assessed by monitoring the generation of vimentin cleavage products by immunoblot analysis. B) Proteolytic processing of vimentin at later stages of infection is dependent on CPAF. The bottom panel represents an increased exposure (OE) of the middle panel. FL: full-length vimentin; C: cleavage product C) LDH release by cells infected with wild type and CPAF-deficient C. trachomatis strains. The supernatants of infected cells were collected at the indicated time points and the amount of LDH release was measured and compared to total LDH levels. Error bars represent standard deviation, n=3.
    Tnex Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .

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

    Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

    doi: 10.4049/jimmunol.0803980

    Figure Lengend Snippet: Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .

    Article Snippet: After harvesting the cells, they were resuspended in PBS with Complete, EDTA-Free Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals).

    Techniques: Isolation, Western Blot, Mass Spectrometry, Sequencing, Transformation Assay, Expressing, Plasmid Preparation, Flow Cytometry, Recombinant, Cytometry, Incubation

    Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

    Journal: Journal of molecular biology

    Article Title: Proteomic Analysis of Ribosomes: Translational Control of mRNA populations by Glycogen Synthase GYS1

    doi: 10.1016/j.jmb.2011.04.064

    Figure Lengend Snippet: Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

    Article Snippet: For preparation of total cell lysates, HeLa cells were washed and harvested in cold PBS, followed by lysis in RIPA buffer (150 mM NaCl, 1mM EDTA, 100 mM Tris-HCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing EDTA-free complete protease inhibitor cocktail (Roche).

    Techniques: Quantitation Assay, Transfection, Polyacrylamide Gel Electrophoresis, Western Blot, Plasmid Preparation

    The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

    Journal: Nucleic Acids Research

    Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    doi: 10.1093/nar/gku692

    Figure Lengend Snippet: The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

    Article Snippet: For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

    Techniques: Mutagenesis, Binding Assay, Proximity Ligation Assay, Labeling, Confocal Microscopy, Immunoprecipitation, Nuclear Magnetic Resonance, Concentration Assay, SYBR Green Assay, Staining

    RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

    Journal: Nucleic Acids Research

    Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    doi: 10.1093/nar/gku692

    Figure Lengend Snippet: RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

    Article Snippet: For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

    Techniques: In Vitro, In Vivo, Clone Assay, Isolation, Immunoprecipitation, Incubation, Ligation, Proximity Ligation Assay, Labeling, Confocal Microscopy

    Evidence for CPAF mediated processing of vimentin in intact cells A) CPAF is not active in 1% SDS buffer. HeLa cell lysates prepared under denaturing conditions in 1% SDS buffer were incubated with the 0.5, 2.5, and 5 μg recombinant CPAF (rCPAF) for 20 minutes at 37 ° C. As a positive control, rCPAF was also incubated with HeLa lysates prepared under non-denaturing conditions in TNEX buffer. Where indicated, 100 μM CPAF inhibitor peptide (CIP) was included as a control. CPAF activity was assessed by monitoring the generation of vimentin cleavage products by immunoblot analysis. B) Proteolytic processing of vimentin at later stages of infection is dependent on CPAF. The bottom panel represents an increased exposure (OE) of the middle panel. FL: full-length vimentin; C: cleavage product C) LDH release by cells infected with wild type and CPAF-deficient C. trachomatis strains. The supernatants of infected cells were collected at the indicated time points and the amount of LDH release was measured and compared to total LDH levels. Error bars represent standard deviation, n=3.

    Journal: Pathogens and disease

    Article Title: REASSESSING THE ROLE OF THE SECRETED PROTEASE CPAF IN CHLAMYDIA TRACHOMATIS INFECTION THROUGH GENETIC APPROACHES

    doi: 10.1111/2049-632X.12179

    Figure Lengend Snippet: Evidence for CPAF mediated processing of vimentin in intact cells A) CPAF is not active in 1% SDS buffer. HeLa cell lysates prepared under denaturing conditions in 1% SDS buffer were incubated with the 0.5, 2.5, and 5 μg recombinant CPAF (rCPAF) for 20 minutes at 37 ° C. As a positive control, rCPAF was also incubated with HeLa lysates prepared under non-denaturing conditions in TNEX buffer. Where indicated, 100 μM CPAF inhibitor peptide (CIP) was included as a control. CPAF activity was assessed by monitoring the generation of vimentin cleavage products by immunoblot analysis. B) Proteolytic processing of vimentin at later stages of infection is dependent on CPAF. The bottom panel represents an increased exposure (OE) of the middle panel. FL: full-length vimentin; C: cleavage product C) LDH release by cells infected with wild type and CPAF-deficient C. trachomatis strains. The supernatants of infected cells were collected at the indicated time points and the amount of LDH release was measured and compared to total LDH levels. Error bars represent standard deviation, n=3.

    Article Snippet: Crude protein extracts were generated from HeLa cells by rinsing monolayers with PBS, adding ice-cold TNEX buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, complete protease inhibitor cocktail (Roche)), incubating at 4 ° C for 10 minutes and then clarifying the lysate by centrifugation (10,000 × g , 15 minutes, 4 ° C).

    Techniques: Incubation, Recombinant, Positive Control, Activity Assay, Infection, Standard Deviation