cacl2 Roche Search Results


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  • 90
    Roche m cacl2
    M Cacl2, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cacl2  (Roche)
    97
    Roche cacl2
    Cacl2, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 3898 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche collagenase a
    Collagenase A, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 5393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cacl2
    Cacl2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15309 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche collagenase d
    Collagenase D, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 10922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Roche binding buffer
    Binding Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 3456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche lysis buffer
    Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 103639 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche buffer a
    Buffer A, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 11218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche complete protease inhibitor
    Complete Protease Inhibitor, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 10600 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Roche cacl2 competent escherichia coli mc1061
    Cacl2 Competent Escherichia Coli Mc1061, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche chaps buffer
    Chaps Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche dispase ii
    Dispase Ii, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 5250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Roche hepes buffer
    Hepes Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 1281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche hypotonic lysis buffer
    Hypotonic Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche proteinase k buffer
    Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after <t>proteinase</t> K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P
    Proteinase K Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Roche sucrose buffer
    Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after <t>proteinase</t> K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P
    Sucrose Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Roche collagenase b dispase ii cacl2 solution
    Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after <t>proteinase</t> K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P
    Collagenase B Dispase Ii Cacl2 Solution, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche liberase tm solution
    Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after <t>proteinase</t> K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P
    Liberase Tm Solution, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche np 40 lysis buffer
    Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after <t>proteinase</t> K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P
    Np 40 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 3422 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche ip buffer
    Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) <t>Immunoprecipitation</t> assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand
    Ip Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 3519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    Roche sodium deoxycholate 1x edta free protease inhibitor
    Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) <t>Immunoprecipitation</t> assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand
    Sodium Deoxycholate 1x Edta Free Protease Inhibitor, supplied by Roche, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Roche deciliation medium
    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine <t>deciliation.</t> Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .
    Deciliation Medium, supplied by Roche, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche complete mini edta free protease inhibitor tablet
    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine <t>deciliation.</t> Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .
    Complete Mini Edta Free Protease Inhibitor Tablet, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Roche annexin v solution
    Analysis of PR1 cells viability after estrogen deprival and restimulation or antiestrogen treatment. (A) Representative cytofluorimetric profile, cells growing in medium containing 10% FCS medium were analyzed before (top) or after treatment with ICI 182,780 (10 -6  M; bottom). M1 indicate the pre-G 1  fraction of dead cells. (B) Kinetics of induction of cell death by ICI 182,780 treatment of PR1 cells. (C) Kinetics of induction of PR1 cell death upon culture in estrogen-free medium (C), without or with ICI 182,780 (ICI), and reversal by stimulation with E2. (D) Cells were maintained in estrogen-free medium (C) for 2 d, before treatment with E2 10 -9  M or ICI 10 -8  M for 24 h and then were analyzed for Annexin V/BOBO-1 double staining by fluorescence microscopy. FCS, cells were maintained in medium containing 10% fetal bovine serum throughout the experiment. The data shown (± SD) are representative of multiple determinations carried out in replicate.
    Annexin V Solution, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Roche annexin buffer
    PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with <t>annexin</t> V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.
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    Roche dnase i digestion buffer
    Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes ( redD and rlpA ) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1 ; qrt-PCR is described elsewhere ( 6 ). <t>DNase</t> I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).
    Dnase I Digestion Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 81/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after proteinase K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P

    Journal: PLoS ONE

    Article Title: C-Terminal ?9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

    doi: 10.1371/journal.pone.0077032

    Figure Lengend Snippet: Trafficking assay of LQT2 mutants located within β9-strand. (A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after proteinase K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V 0.5 (open symbols) and 3 s isochronal deactivation V 0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate P

    Article Snippet: Proteinase K digestion assays HEK293 cells expressing WT or mutant hERG1a constructs were washed with PBS and incubated in proteinase K buffer (in mM, HEPES 10, NaCl 150, CaCl2 2, KCl 10, pH 7.4) with 200 µg/mL proteinase K (Roche, Castle Hill, NSW, Australia) for 45 min at 37°C.

    Techniques: Western Blot, Mutagenesis, Expressing, Incubation, Activation Assay

    Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand

    Journal: Cell Death & Disease

    Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

    doi: 10.1038/cddis.2014.193

    Figure Lengend Snippet: Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand

    Article Snippet: Immunoprecipitation, mass spectrometry and western blot Thirty-six hours after transient transfection, HEK293T cells were lysed on ice in immunoprecipitation buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 2 mM CaCl2 , pH 7.4) with a protease inhibitor cocktail (Roche, Basel, Switzerland).

    Techniques: Binding Assay, Immunoprecipitation, Expressing, SDS Page, Mass Spectrometry, Transfection

    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine deciliation. Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .

    Journal: Methods in enzymology

    Article Title: Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila

    doi: 10.1016/B978-0-12-397944-5.00013-4

    Figure Lengend Snippet: A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine deciliation. Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .

    Article Snippet: Collect cells by centrifugation (1700 × g 3 min; swinging bucket rotor, 50 ml conical tubes), wash once with 10 mM Tris-HCl pH 7.5 and gently suspend in 20 ml of the deciliation medium (10 mM Tris-HCl pH 7.4, 50 mM sucrose, 10 mM CaCl2 , protease inhibitors (Complete, Roche)) in a 250 ml flask.

    Techniques: Purification, Staining

    Analysis of PR1 cells viability after estrogen deprival and restimulation or antiestrogen treatment. (A) Representative cytofluorimetric profile, cells growing in medium containing 10% FCS medium were analyzed before (top) or after treatment with ICI 182,780 (10 -6  M; bottom). M1 indicate the pre-G 1  fraction of dead cells. (B) Kinetics of induction of cell death by ICI 182,780 treatment of PR1 cells. (C) Kinetics of induction of PR1 cell death upon culture in estrogen-free medium (C), without or with ICI 182,780 (ICI), and reversal by stimulation with E2. (D) Cells were maintained in estrogen-free medium (C) for 2 d, before treatment with E2 10 -9  M or ICI 10 -8  M for 24 h and then were analyzed for Annexin V/BOBO-1 double staining by fluorescence microscopy. FCS, cells were maintained in medium containing 10% fetal bovine serum throughout the experiment. The data shown (± SD) are representative of multiple determinations carried out in replicate.

    Journal: Molecular Biology of the Cell

    Article Title: Distinct Signaling Pathways Mediate Stimulation of Cell Cycle Progression and Prevention of Apoptotic Cell Death by Estrogen in Rat Pituitary Tumor PR1 Cells

    doi: 10.1091/mbc.E03-05-0303

    Figure Lengend Snippet: Analysis of PR1 cells viability after estrogen deprival and restimulation or antiestrogen treatment. (A) Representative cytofluorimetric profile, cells growing in medium containing 10% FCS medium were analyzed before (top) or after treatment with ICI 182,780 (10 -6 M; bottom). M1 indicate the pre-G 1 fraction of dead cells. (B) Kinetics of induction of cell death by ICI 182,780 treatment of PR1 cells. (C) Kinetics of induction of PR1 cell death upon culture in estrogen-free medium (C), without or with ICI 182,780 (ICI), and reversal by stimulation with E2. (D) Cells were maintained in estrogen-free medium (C) for 2 d, before treatment with E2 10 -9 M or ICI 10 -8 M for 24 h and then were analyzed for Annexin V/BOBO-1 double staining by fluorescence microscopy. FCS, cells were maintained in medium containing 10% fetal bovine serum throughout the experiment. The data shown (± SD) are representative of multiple determinations carried out in replicate.

    Article Snippet: For Annexin V-BOBO-1 labeling, cells were plated (30,000 cells/well) in 24-well dishes and, after stimulation, the medium was removed and cells were incubated in the Annexin V solution (10 mM HEPES, 140 mM NaCl and CaCl2 , and 1 μg/ml Annexin V-Alexa-568; Roche Diagnostics, Mannheim, Germany), 1 μg/ml BOBO-1 (Molecular Probes).

    Techniques: Double Staining, Fluorescence, Microscopy

    PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with annexin V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with annexin V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Incubation, Labeling, Flow Cytometry, Cytometry, Immunoprecipitation, Activity Assay, Colorimetric Assay

    Effect of stable transfection of EBNA-LP on apoptosis. Ramos cells were cotransfected with EBNA-LP expression vectors and the pSG5(Neo R ) vector, which carries the neomycin resistance gene. The cells were grown in selection medium (complete RPMI supplemented with 16 mg/ml neomycin) for 4 weeks, and the clones were then tested. (A) Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. Molecular sizes are indicated to the right. (B) Stable transfectants were treated with VT-1 (5 ng/ml) for 16 h, labeled with annexin V-FITC and PI, and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars shown the standard deviations.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: Effect of stable transfection of EBNA-LP on apoptosis. Ramos cells were cotransfected with EBNA-LP expression vectors and the pSG5(Neo R ) vector, which carries the neomycin resistance gene. The cells were grown in selection medium (complete RPMI supplemented with 16 mg/ml neomycin) for 4 weeks, and the clones were then tested. (A) Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. Molecular sizes are indicated to the right. (B) Stable transfectants were treated with VT-1 (5 ng/ml) for 16 h, labeled with annexin V-FITC and PI, and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars shown the standard deviations.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Selection, Clone Assay, Electrophoresis, Labeling, Flow Cytometry, Cytometry, Inhibition, Transfection

    Infection with the P3HR1 strain of EBV protects BL cells against VT-1- and staurosporine-induced apoptosis. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: Infection with the P3HR1 strain of EBV protects BL cells against VT-1- and staurosporine-induced apoptosis. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Infection, Incubation, Labeling, Flow Cytometry, Cytometry, Electrophoresis

    VT-1 and staurosporine induce caspase-dependent apoptotic cell death in some, but not all, BL cell lines. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: VT-1 and staurosporine induce caspase-dependent apoptotic cell death in some, but not all, BL cell lines. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Incubation, Labeling, Flow Cytometry, Cytometry, Electrophoresis

    Impaired survival and proliferation of PKCβ-deficient B cells in vitro. (A) Accelerated cell death of PKCβ −/− B cells cultured without stimulation. Percentage of cell viability of purified splenic B cells is plotted for wild-type (129/Sv) control (open circles) and PKCβ −/− (filled circles) B cells. Each dot represents cells isolated from an individual mouse. (B) Impaired proliferative responses of PKCβ −/− B cells in vitro in the absence of IL-4 survival signal. Purified splenic B cells of wild-type (left column) or PKCβ −/− mice (right column) were labeled with CFSE and incubated for 3 d in the presence or absence of the indicated stimuli (see Materials and Methods for details). The thick line indicates the level of CFSE florescence in cells upon stimulation, whereas the thin line indicates the CFSE label of nonstimulated cells. Reduction in the CFSE fluorescence is proportional to the number of cell divisions. (C) IL-4 promotes survival of both wild-type and PKCβ −/− B cells. FACS ® analysis of Annexin-V in combination of 7AAD staining of wild-type (left column) and PKCβ −/− (right column) B cells cultured in complete RPMI with or without IL-4. Note the increase of live cells annexin-V and 7AAD negative in the presence of IL-4. Numbers indicate percentages of gated cells.

    Journal: The Journal of Experimental Medicine

    Article Title: Protein Kinase C ? Controls Nuclear Factor ?B Activation in B Cells Through Selective Regulation of the I?B Kinase ?

    doi: 10.1084/jem.20020408

    Figure Lengend Snippet: Impaired survival and proliferation of PKCβ-deficient B cells in vitro. (A) Accelerated cell death of PKCβ −/− B cells cultured without stimulation. Percentage of cell viability of purified splenic B cells is plotted for wild-type (129/Sv) control (open circles) and PKCβ −/− (filled circles) B cells. Each dot represents cells isolated from an individual mouse. (B) Impaired proliferative responses of PKCβ −/− B cells in vitro in the absence of IL-4 survival signal. Purified splenic B cells of wild-type (left column) or PKCβ −/− mice (right column) were labeled with CFSE and incubated for 3 d in the presence or absence of the indicated stimuli (see Materials and Methods for details). The thick line indicates the level of CFSE florescence in cells upon stimulation, whereas the thin line indicates the CFSE label of nonstimulated cells. Reduction in the CFSE fluorescence is proportional to the number of cell divisions. (C) IL-4 promotes survival of both wild-type and PKCβ −/− B cells. FACS ® analysis of Annexin-V in combination of 7AAD staining of wild-type (left column) and PKCβ −/− (right column) B cells cultured in complete RPMI with or without IL-4. Note the increase of live cells annexin-V and 7AAD negative in the presence of IL-4. Numbers indicate percentages of gated cells.

    Article Snippet: Cells were washed once with ice-cold Annexin V binding buffer (10 mM Hepes, pH 7.5, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) and cell pellets were stained with Annexin V (Roche) and 7-aminoactinomycin D (7AAD; Sigma-Aldrich) as described ( ).

    Techniques: In Vitro, Cell Culture, Purification, Isolation, Mouse Assay, Labeling, Incubation, Fluorescence, FACS, Staining

    Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes ( redD and rlpA ) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1 ; qrt-PCR is described elsewhere ( 6 ). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes ( redD and rlpA ) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1 ; qrt-PCR is described elsewhere ( 6 ). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: Quantitative RT-PCR, In Vivo

    Transcriptionally regulated genes in S.coelicolor show a positive correlation between DNase I-sensitivity and level of expression. qrt-PCR was used to measure both relative DNase I sensitivity and RNA abundance ( Figure 1 , lower) for each of the twelve genes used in this study at the three timepoints described. Expression data is relative to the value of act II-orf4 gene at T3 timepoint. Each point is the average of at least three independent measurements with standard deviations shown as dotted lines.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: Transcriptionally regulated genes in S.coelicolor show a positive correlation between DNase I-sensitivity and level of expression. qrt-PCR was used to measure both relative DNase I sensitivity and RNA abundance ( Figure 1 , lower) for each of the twelve genes used in this study at the three timepoints described. Expression data is relative to the value of act II-orf4 gene at T3 timepoint. Each point is the average of at least three independent measurements with standard deviations shown as dotted lines.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: Expressing, Quantitative RT-PCR, Activated Clotting Time Assay

    In vivo DNase I digestion of the S.coelicolor genome results in fractionation according to transcriptional activity. ( A ) HCHO-crosslinked DNA–protein complexes from S.coelicolor collected at T1 (shown as an example with the triangle representing increasing amounts of DNase I) and T3 were digested in vivo with increasing amounts of DNase I, and the nucleoprotein complexes separated by 0.7% agarose gel electrophoresis. DNA was extracted from the soluble fraction, defined as that resolved by the gel and indicated by a box, and used as a probe in the subsequent slot-blot experiment. ( B ) DNA and RNA samples were labeled with DIG-dUTP and used as probes in slot-blot hybridizations with 12 ∼500 bp PCR products corresponding to the panel of genes used in the experiment. At each time point, the RNA and DNA probes detect the same targets, demonstrating that in vivo DNase I treatment had fractionated the genome based on transcriptional activity. Different targets were detected by samples from time points T1 and T3, consistent with the transcriptional programme changing as S.coelicolor enters stationary phase.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: In vivo DNase I digestion of the S.coelicolor genome results in fractionation according to transcriptional activity. ( A ) HCHO-crosslinked DNA–protein complexes from S.coelicolor collected at T1 (shown as an example with the triangle representing increasing amounts of DNase I) and T3 were digested in vivo with increasing amounts of DNase I, and the nucleoprotein complexes separated by 0.7% agarose gel electrophoresis. DNA was extracted from the soluble fraction, defined as that resolved by the gel and indicated by a box, and used as a probe in the subsequent slot-blot experiment. ( B ) DNA and RNA samples were labeled with DIG-dUTP and used as probes in slot-blot hybridizations with 12 ∼500 bp PCR products corresponding to the panel of genes used in the experiment. At each time point, the RNA and DNA probes detect the same targets, demonstrating that in vivo DNase I treatment had fractionated the genome based on transcriptional activity. Different targets were detected by samples from time points T1 and T3, consistent with the transcriptional programme changing as S.coelicolor enters stationary phase.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: In Vivo, Fractionation, Activity Assay, Agarose Gel Electrophoresis, Dot Blot, Labeling, Polymerase Chain Reaction

    Gene expression during growth of  S.coelicolor  strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: Gene expression during growth of S.coelicolor strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: Expressing, Activated Clotting Time Assay, Quantitative RT-PCR

    In vivo DNase I sensitivity determined by Southern hybridization analysis. DNA harvested from mycelium taken from each time point was treated with increasing amounts of DNase I, digested to completion with restriction enzymes and used in Southern hybridization assays. Blots were hybridized with mixtures of probes for ( A ) rbnH , redD , rlpA and sti1 and ( B ) act II-orf4, afsQ2 , hrdB and rpmG3 . The positions of the genomic bands for each gene are indicated, with the amount of DNase I used increasing from left to right.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: In vivo DNase I sensitivity determined by Southern hybridization analysis. DNA harvested from mycelium taken from each time point was treated with increasing amounts of DNase I, digested to completion with restriction enzymes and used in Southern hybridization assays. Blots were hybridized with mixtures of probes for ( A ) rbnH , redD , rlpA and sti1 and ( B ) act II-orf4, afsQ2 , hrdB and rpmG3 . The positions of the genomic bands for each gene are indicated, with the amount of DNase I used increasing from left to right.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: In Vivo, Hybridization, Activated Clotting Time Assay