cacl2  (Roche)


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    Structured Review

    Roche cacl2
    Cacl2, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lysis:

    Article Title: DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs
    Article Snippet: .. Pellets were resuspended in native lysis buffer (50 mM NaH2 PO4 pH 8.0; 300 mM NaCl and 10 mM imidazole) supplemented with 1 mg/ml Lysozyme (Sigma), 30 U/ml Nuclease S7 (Roche), 1 mM CaCl2 , 1% Triton X-100 and protease inhibitor cocktail (Roche). ..

    Article Title: Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis.
    Article Snippet: .. In the plant nucleus, the majority of cellular DNA content is stored and maintained. .. In the plant nucleus, the majority of cellular DNA content is stored and maintained.

    Article Title: Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System
    Article Snippet: .. A total of 1 × 108 bacteria of each culture were pelleted at 8,000 rpm for 5 min, resuspended in 190 μl of lysis buffer (60 mM Tris [pH 8], 10 mM MgCl2 , 50 μM CaCl2 , 50 μg/ml DNase, 200 μg/ml lysozyme, protease inhibitor cocktail [Roche]) and incubated on ice for 45 min. ..

    Incubation:

    Article Title: Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System
    Article Snippet: .. A total of 1 × 108 bacteria of each culture were pelleted at 8,000 rpm for 5 min, resuspended in 190 μl of lysis buffer (60 mM Tris [pH 8], 10 mM MgCl2 , 50 μM CaCl2 , 50 μg/ml DNase, 200 μg/ml lysozyme, protease inhibitor cocktail [Roche]) and incubated on ice for 45 min. ..

    Protease Inhibitor:

    Article Title: DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs
    Article Snippet: .. Pellets were resuspended in native lysis buffer (50 mM NaH2 PO4 pH 8.0; 300 mM NaCl and 10 mM imidazole) supplemented with 1 mg/ml Lysozyme (Sigma), 30 U/ml Nuclease S7 (Roche), 1 mM CaCl2 , 1% Triton X-100 and protease inhibitor cocktail (Roche). ..

    Article Title: ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding
    Article Snippet: .. Protein lysates were prepared in 0.5% NP-40 in a background buffer of either 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA or 50 mM HEPES-KOH (pH 6.8), 150 mM potassium acetate (KOAc), 2 mM magnesium acetate (MgOAc), 1 mM CaCl2 , 15% glycerol, 1× Roche EDTA-free protease inhibitor cocktail. ..

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: .. Briefly, A2780 cells (107 cells) before and after 5-aza-CdR, G9Ai, or combination treatment were harvested and washed twice and resuspended in ice-cold Buffer N (15 mM Tris pH7.5, 15 mM NaCl, 60 mM KCl, 8.5%(w/v) Surcose, 5 mM MgCl2 , 1 mM CaCl2 , 1 mM DTT, 200 μM PMSF, 1X cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche)). .. To prepare nuclei, cells were lysed in 1 mL Lysis Buffer (Buffer N supplemented with 0.3% NP-40 substitute (Sigma)) for 10 min at 4°C, and nuclei were collected by centrifugation (500 × g for 5 min at 4°C), resuspended in 1 mL of Buffer N, then sedimented through 7.5 mL sucrose cushion (10 mM HEPES pH7.9, 30%(w/v) sucrose, 1.5 mM MgCl2 and centrifuged 13000 × g using Sorvall swinging bucket for 12 min at 4°C).

    Article Title: Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System
    Article Snippet: .. A total of 1 × 108 bacteria of each culture were pelleted at 8,000 rpm for 5 min, resuspended in 190 μl of lysis buffer (60 mM Tris [pH 8], 10 mM MgCl2 , 50 μM CaCl2 , 50 μg/ml DNase, 200 μg/ml lysozyme, protease inhibitor cocktail [Roche]) and incubated on ice for 45 min. ..

    Immunoprecipitation:

    Article Title: Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition
    Article Snippet: .. Immunoprecipitation Human embryonic kidney 293T or human osteosarcoma U-2 OS cells were treated as indicated, washed 2 times with ice-cold PBS and resuspended in cytoplasmic extraction buffer [10 mM Tris·HCl (pH 7.9), 0.34 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 20 mM NaF, 10 mM β-glycerophosphate, 0.2 mM Na3VO4, 0.5% Nonidet P-40 and protease inhibitors (Roche)] for 10 min at 4°C. .. Intact nuclei were pelleted by low-speed centrifugation, washed with cytoplasmic lysis buffer (without Nonidet P-40), lysed in nuclear lysis buffer [20 mM HEPES (pH 7.9), 150 mM KCl, 1.5 mM MgCl2, 20 mM NaF, 10 mM β-glycerophosphate, 0.2 mM Na3 VO4 10% glycerol, 0.5% Nonidet P-40, and protease inhibitors] by homogenization and DNA and RNA in the suspension were digested with 50 U/μl Benzonase (Sigma) at 4°C for 1 hour.

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    Roche dnase i cocktail
    I-loops are induced by single-strand damage at telomeric repeats (related to Figure 5 ). A. 2D-gel analysis showing that the t-circle arc is strongly induced by formation of nicks and gaps at human telomeres. Nuclei prepared from HeLa 1.3 cells (this HeLa clone has long telomeres around, ~20 kb in average) were incubated with either 0; 0.5; 1 or 2.5 μg/ml of <t>DNase</t> I for 8 minutes at RT. Nuclei prepared from HTC75 cells (with telomeres around 4 kb in average) and from HeLa 204 cells (with telomeres around, ~2 kb in average) were incubated with either 0; 5; 10 or 20 μg/ml of DNase I for 8 minutes at RT. The nuclei were then processed for 2D-gel analysis, as described in Figure 5A . B. 2D-gel analysis showing that DNase I treatment does not induce the t-circle arc in the bulk DNA or at the BamHI repeats. In a similar experiment as the one described in Figure 5A , nuclei were treated with 2.5 μg/ml of DNase I. Genomic was digested with BglI, split in two and separated on 2D-gels, in duplicate. After blotting, one membrane was hybridized with a probe recognizing the TTAGGG repeats, while the other with a probe recognizing the mouse BamHI repeats. The ethidium bromide staining of one of the second-dimension gels is shown at the bottom. C. 2D-gel analysis showing that DNase I treatment on isolated DNA does not induce the t-circle arc in the bulk DNA or at the BamHI repeats. DNA from the same experiment shown in Figure 5B , was digested with KpnI, split in two and then separated in 2D-gels, in duplicate. After blotting, one membrane was hybridized with a probe recognizing the TTAGGG repeats and the other with a probe recognizing the mouse BamHI repeats. The ethidium bromide staining of one of the second-dimension gels is shown at the bottom.
    Dnase I Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    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: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche immunoprecipitation buffer
    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of <t>immunoprecipitation</t> eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    I-loops are induced by single-strand damage at telomeric repeats (related to Figure 5 ). A. 2D-gel analysis showing that the t-circle arc is strongly induced by formation of nicks and gaps at human telomeres. Nuclei prepared from HeLa 1.3 cells (this HeLa clone has long telomeres around, ~20 kb in average) were incubated with either 0; 0.5; 1 or 2.5 μg/ml of DNase I for 8 minutes at RT. Nuclei prepared from HTC75 cells (with telomeres around 4 kb in average) and from HeLa 204 cells (with telomeres around, ~2 kb in average) were incubated with either 0; 5; 10 or 20 μg/ml of DNase I for 8 minutes at RT. The nuclei were then processed for 2D-gel analysis, as described in Figure 5A . B. 2D-gel analysis showing that DNase I treatment does not induce the t-circle arc in the bulk DNA or at the BamHI repeats. In a similar experiment as the one described in Figure 5A , nuclei were treated with 2.5 μg/ml of DNase I. Genomic was digested with BglI, split in two and separated on 2D-gels, in duplicate. After blotting, one membrane was hybridized with a probe recognizing the TTAGGG repeats, while the other with a probe recognizing the mouse BamHI repeats. The ethidium bromide staining of one of the second-dimension gels is shown at the bottom. C. 2D-gel analysis showing that DNase I treatment on isolated DNA does not induce the t-circle arc in the bulk DNA or at the BamHI repeats. DNA from the same experiment shown in Figure 5B , was digested with KpnI, split in two and then separated in 2D-gels, in duplicate. After blotting, one membrane was hybridized with a probe recognizing the TTAGGG repeats and the other with a probe recognizing the mouse BamHI repeats. The ethidium bromide staining of one of the second-dimension gels is shown at the bottom.

    Journal: bioRxiv

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1101/2020.01.29.924951

    Figure Lengend Snippet: I-loops are induced by single-strand damage at telomeric repeats (related to Figure 5 ). A. 2D-gel analysis showing that the t-circle arc is strongly induced by formation of nicks and gaps at human telomeres. Nuclei prepared from HeLa 1.3 cells (this HeLa clone has long telomeres around, ~20 kb in average) were incubated with either 0; 0.5; 1 or 2.5 μg/ml of DNase I for 8 minutes at RT. Nuclei prepared from HTC75 cells (with telomeres around 4 kb in average) and from HeLa 204 cells (with telomeres around, ~2 kb in average) were incubated with either 0; 5; 10 or 20 μg/ml of DNase I for 8 minutes at RT. The nuclei were then processed for 2D-gel analysis, as described in Figure 5A . B. 2D-gel analysis showing that DNase I treatment does not induce the t-circle arc in the bulk DNA or at the BamHI repeats. In a similar experiment as the one described in Figure 5A , nuclei were treated with 2.5 μg/ml of DNase I. Genomic was digested with BglI, split in two and separated on 2D-gels, in duplicate. After blotting, one membrane was hybridized with a probe recognizing the TTAGGG repeats, while the other with a probe recognizing the mouse BamHI repeats. The ethidium bromide staining of one of the second-dimension gels is shown at the bottom. C. 2D-gel analysis showing that DNase I treatment on isolated DNA does not induce the t-circle arc in the bulk DNA or at the BamHI repeats. DNA from the same experiment shown in Figure 5B , was digested with KpnI, split in two and then separated in 2D-gels, in duplicate. After blotting, one membrane was hybridized with a probe recognizing the TTAGGG repeats and the other with a probe recognizing the mouse BamHI repeats. The ethidium bromide staining of one of the second-dimension gels is shown at the bottom.

    Article Snippet: For the DNase I treatment, 1 volume of nuclei suspension was mixed with 1 volume of DNase I cocktail (NWB supplemented with CaCl2 2 mM, BSA 100 μg/ml, and twice the indicated concentration of DNase I (Roche 10104159001) and incubated for 8 minutes at RT.

    Techniques: Two-Dimensional Gel Electrophoresis, Incubation, Staining, Isolation

    I-loops are a substrate for the generation of extrachromosomal telomeric circles. A.  Model for the formation of i-loops, in the presence of short single-strand gaps on opposite telomeric strands (i.e. one gap on the G-strand and one on the C-strand of the same molecule). Exposed complementary DNA can come in close proximity by looping and undergo base pairing (step 1). Plectonemic pairing can occur simply by strand rotation, resulting in the formation of an i-loop that will resemble a DNA knot (step 2). The loop junction could branch migrate as a hemicatenane (step 3) that could be transformed in a double Holliday junction by the pairing of the opposite strands (step 4). B.  Model for the formation of i-loops, in the presence of short single-strand gaps on the same telomeric strand (i.e. both gaps on the G or on the C strand of the same molecule). The gaps can come in close proximity by DNA looping (step 1) and promote an exchange of the complementary strands (step 2) resulting in an intramolecular loop with a single Holliday junction at the base (step 3), that can undergo branch migration (step 4). C.  Model for the generation of the telomeric circles via the excision of i-loops. An i-loop with a Holliday Junction at the base becomes a substrate for Holliday Junction resolvases. Cleavage on the horizontal axis of the image (orange arrows) will result in the excision of the loop as a circle and telomere loss. Note that the excised circle, would contain a nick, resulting from the HJ resolution and a single-stranded gap (one of the original gaps that induced formation of the i-loop). D.  Schematic representation of the experimental procedure used to test the model shown in (C). Isolated DNA was nicked with low concentrations of DNase I, which induces the formation of i-loops at telomeres. The reaction was stopped, DNA was extracted and incubated for 30 minutes at 37°C with a HeLa nuclear extract in the presence of Mg++ and ATP, to allow HJ resolution. The DNA was then purified and the presence of telomeric circles was assayed with the C-circle assay. E.  Dot blot analysis of the circle assay of the experiment described in (D). The DNA from the C-circle assay was blotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. A strong C-circle signal accumulates only in the combined treatment nicking and incubation with the extract. Quantification of the C-circle signal from 3 independent experiments as the one described in (D). The signal is reported relative to the untreated sample (no DNase I, no extract) which was set to 1. P value was derived from unpaired, two-tailed, Student’s t-test.

    Journal: bioRxiv

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1101/2020.01.29.924951

    Figure Lengend Snippet: I-loops are a substrate for the generation of extrachromosomal telomeric circles. A. Model for the formation of i-loops, in the presence of short single-strand gaps on opposite telomeric strands (i.e. one gap on the G-strand and one on the C-strand of the same molecule). Exposed complementary DNA can come in close proximity by looping and undergo base pairing (step 1). Plectonemic pairing can occur simply by strand rotation, resulting in the formation of an i-loop that will resemble a DNA knot (step 2). The loop junction could branch migrate as a hemicatenane (step 3) that could be transformed in a double Holliday junction by the pairing of the opposite strands (step 4). B. Model for the formation of i-loops, in the presence of short single-strand gaps on the same telomeric strand (i.e. both gaps on the G or on the C strand of the same molecule). The gaps can come in close proximity by DNA looping (step 1) and promote an exchange of the complementary strands (step 2) resulting in an intramolecular loop with a single Holliday junction at the base (step 3), that can undergo branch migration (step 4). C. Model for the generation of the telomeric circles via the excision of i-loops. An i-loop with a Holliday Junction at the base becomes a substrate for Holliday Junction resolvases. Cleavage on the horizontal axis of the image (orange arrows) will result in the excision of the loop as a circle and telomere loss. Note that the excised circle, would contain a nick, resulting from the HJ resolution and a single-stranded gap (one of the original gaps that induced formation of the i-loop). D. Schematic representation of the experimental procedure used to test the model shown in (C). Isolated DNA was nicked with low concentrations of DNase I, which induces the formation of i-loops at telomeres. The reaction was stopped, DNA was extracted and incubated for 30 minutes at 37°C with a HeLa nuclear extract in the presence of Mg++ and ATP, to allow HJ resolution. The DNA was then purified and the presence of telomeric circles was assayed with the C-circle assay. E. Dot blot analysis of the circle assay of the experiment described in (D). The DNA from the C-circle assay was blotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. A strong C-circle signal accumulates only in the combined treatment nicking and incubation with the extract. Quantification of the C-circle signal from 3 independent experiments as the one described in (D). The signal is reported relative to the untreated sample (no DNase I, no extract) which was set to 1. P value was derived from unpaired, two-tailed, Student’s t-test.

    Article Snippet: For the DNase I treatment, 1 volume of nuclei suspension was mixed with 1 volume of DNase I cocktail (NWB supplemented with CaCl2 2 mM, BSA 100 μg/ml, and twice the indicated concentration of DNase I (Roche 10104159001) and incubated for 8 minutes at RT.

    Techniques: Transformation Assay, Migration, Isolation, Incubation, Purification, Dot Blot, Derivative Assay, Two Tailed Test

    I-loops are induced by single-strand damage at telomeric repeats (see also Figure S5 ). A. 2D-gel analysis showing that the t-circle arc can be strongly induced by formation of nicks and gaps at telomeres. MEFs nuclei were incubated with either 0; 1; 2.5 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped and the genomic DNA was isolated. 5 μg were digested with AluI and MboI and separated on 2D-gels. The gels were blotted on a membrane and hybridized with a TTAGGG repeats probe. The ratio of the telomeric signal in the t-circle arc (yellow arrows) and in the linears (black arrows), is reported relative to the untreated sample, which was arbitrarily set to 100. B. 2D-gel analysis showing that the t-circle arc can form spontaneously, in the presence of nicks and gaps at the telomeric repeats. Isolated mouse genomic DNA was incubated with either 0; 0.1; 0.2 or 0.4 μg/ml of DNase I for 8 min at RT. The reaction was stopped, the DNA was extracted with phenol-chloroform, digested with AluI and MboI, separated on 2D-gels, blotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the untreated sample which was arbitrarily set to 100. C. Dot blot showing the enrichment of the telomeric repeats, after the large-scale DNase I treatment. Around 500 × 10 6 SV40-MEFs nuclei were incubated either with 0 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped, genomic DNA was isolated and telomeres were enriched with the procedure described in Figure 1 . The indicated amounts from each enrichment step were spotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The telomeric signal per ng of DNA is reported relative to the non-enriched DNA. D. Accumulation of i-loops at telomeres damaged by DNase I. Telomere-enriched DNA from the experiment described in (C) was analyzed in EM. The percentage of molecules with internal loops is reported. A KpnI-digested bulk DNA control was included for the sample treated with DNase I. E. Examples of molecules with internal loops observed at telomere preparations, from DNase I-treated nuclei. Insets show 2X enlargements of the area inside the yellow rectangles.

    Journal: bioRxiv

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1101/2020.01.29.924951

    Figure Lengend Snippet: I-loops are induced by single-strand damage at telomeric repeats (see also Figure S5 ). A. 2D-gel analysis showing that the t-circle arc can be strongly induced by formation of nicks and gaps at telomeres. MEFs nuclei were incubated with either 0; 1; 2.5 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped and the genomic DNA was isolated. 5 μg were digested with AluI and MboI and separated on 2D-gels. The gels were blotted on a membrane and hybridized with a TTAGGG repeats probe. The ratio of the telomeric signal in the t-circle arc (yellow arrows) and in the linears (black arrows), is reported relative to the untreated sample, which was arbitrarily set to 100. B. 2D-gel analysis showing that the t-circle arc can form spontaneously, in the presence of nicks and gaps at the telomeric repeats. Isolated mouse genomic DNA was incubated with either 0; 0.1; 0.2 or 0.4 μg/ml of DNase I for 8 min at RT. The reaction was stopped, the DNA was extracted with phenol-chloroform, digested with AluI and MboI, separated on 2D-gels, blotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the untreated sample which was arbitrarily set to 100. C. Dot blot showing the enrichment of the telomeric repeats, after the large-scale DNase I treatment. Around 500 × 10 6 SV40-MEFs nuclei were incubated either with 0 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped, genomic DNA was isolated and telomeres were enriched with the procedure described in Figure 1 . The indicated amounts from each enrichment step were spotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The telomeric signal per ng of DNA is reported relative to the non-enriched DNA. D. Accumulation of i-loops at telomeres damaged by DNase I. Telomere-enriched DNA from the experiment described in (C) was analyzed in EM. The percentage of molecules with internal loops is reported. A KpnI-digested bulk DNA control was included for the sample treated with DNase I. E. Examples of molecules with internal loops observed at telomere preparations, from DNase I-treated nuclei. Insets show 2X enlargements of the area inside the yellow rectangles.

    Article Snippet: For the DNase I treatment, 1 volume of nuclei suspension was mixed with 1 volume of DNase I cocktail (NWB supplemented with CaCl2 2 mM, BSA 100 μg/ml, and twice the indicated concentration of DNase I (Roche 10104159001) and incubated for 8 minutes at RT.

    Techniques: Two-Dimensional Gel Electrophoresis, Incubation, Isolation, Dot Blot

    I-loop formation following the induction of DNA damage requires branch migration. A. A preparation of SV40-MEFs nuclei was split in two and one half was psoralen crosslinked on ice (in this prep, DNA branch migration is largely prevented). Then, both preps were treated with DNase I and processed for 2D-gels as described in Figure 5A . The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the untreated and crosslinked sample, which was arbitrarily set to 100. B. 2D-gel analysis showing that the intramolecular loops that accumulate in ALT cells are not affected by psoralen crosslinking. U2OS cells were psoralen crosslinked on ice, then genomic DNA was extracted and processed for 2D-gels as above. The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the crosslinked sample which was arbitrarily set to 100.

    Journal: bioRxiv

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1101/2020.01.29.924951

    Figure Lengend Snippet: I-loop formation following the induction of DNA damage requires branch migration. A. A preparation of SV40-MEFs nuclei was split in two and one half was psoralen crosslinked on ice (in this prep, DNA branch migration is largely prevented). Then, both preps were treated with DNase I and processed for 2D-gels as described in Figure 5A . The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the untreated and crosslinked sample, which was arbitrarily set to 100. B. 2D-gel analysis showing that the intramolecular loops that accumulate in ALT cells are not affected by psoralen crosslinking. U2OS cells were psoralen crosslinked on ice, then genomic DNA was extracted and processed for 2D-gels as above. The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the crosslinked sample which was arbitrarily set to 100.

    Article Snippet: For the DNase I treatment, 1 volume of nuclei suspension was mixed with 1 volume of DNase I cocktail (NWB supplemented with CaCl2 2 mM, BSA 100 μg/ml, and twice the indicated concentration of DNase I (Roche 10104159001) and incubated for 8 minutes at RT.

    Techniques: Migration, Two-Dimensional Gel Electrophoresis

    Composition of the biofilm matrix. a CLSM micrographs of biofilms stained with BOBO-3 (exDNA, Scale bar: 10 µm), Sypro Ruby (proteins, Scale bar: 20 µm), WGA (N-acetylglucosamine or sialic acid residues, Scale bar: 10 µm) and ConA (red/α-D-glucosyl or α-D-mannosyl residues, Scale bar: 10 µm), red, upper line. Bacteria were stained with a CFDA-SE cell-tracker (green, middle line). Merged images are shown in the lower line. b Phase contrast images of biofilms left untreated or treated for 1, 3, 6, 24 and 48 h with proteinase K (1 µg/mL), DNase I (6.25 µg/mL), alginate lyase (125 U/mL) and sodium metaperiodate (5 mM). Scale bar: 500 µm. c Surface based quantification of the area occupied by the biofilm after 48 h of exposure to proteinase K (orange), DNase I (purple), Alginate Lyase (green) and sodium metaperiodate (blue). **** indicates a p value

    Journal: NPJ Biofilms and Microbiomes

    Article Title: The zoonotic pathogen Leptospira interrogans mitigates environmental stress through cyclic-di-GMP-controlled biofilm production

    doi: 10.1038/s41522-020-0134-1

    Figure Lengend Snippet: Composition of the biofilm matrix. a CLSM micrographs of biofilms stained with BOBO-3 (exDNA, Scale bar: 10 µm), Sypro Ruby (proteins, Scale bar: 20 µm), WGA (N-acetylglucosamine or sialic acid residues, Scale bar: 10 µm) and ConA (red/α-D-glucosyl or α-D-mannosyl residues, Scale bar: 10 µm), red, upper line. Bacteria were stained with a CFDA-SE cell-tracker (green, middle line). Merged images are shown in the lower line. b Phase contrast images of biofilms left untreated or treated for 1, 3, 6, 24 and 48 h with proteinase K (1 µg/mL), DNase I (6.25 µg/mL), alginate lyase (125 U/mL) and sodium metaperiodate (5 mM). Scale bar: 500 µm. c Surface based quantification of the area occupied by the biofilm after 48 h of exposure to proteinase K (orange), DNase I (purple), Alginate Lyase (green) and sodium metaperiodate (blue). **** indicates a p value

    Article Snippet: Dissociation of biofilms by chemical or enzymatic treatments WT biofilms were grown as described in the Biofilm Formation section, and after 21 days of incubation, twenty µL of DNase I (6.25–500 µg/mL in 150 mM NaCl, 1 mM CaCl2 ; 10104159001; Roche Diagnostics GmbH, Manheim, Germany), 20 µL of proteinase K (6.25–500 µg/mL in 50 mM Tris-HCl pH 7.5, 1 mM CaCl2; 05323738001; Roche Diagnostics GmbH, Manheim, Germany), 20 µL of alginate lyase (1–200 U in 20 mM Tris, 200 mM NaCl; A-1603-100MG, Sigma Aldrich, St-Louis, MO, USA) and 20 µL of sodium periodate (1–100 mM in 50 mM sodium acetate; S1878-25G, Sigma Aldrich, St-Louis, MO, USA) were added directly to the biofilms.

    Techniques: Confocal Laser Scanning Microscopy, Staining, Whole Genome Amplification

    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

    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Journal: The Journal of Neuroscience

    Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

    doi: 10.1523/JNEUROSCI.2548-14.2015

    Figure Lengend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Article Snippet: For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche).

    Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture