mutant u3 snorna genes  (Millipore)


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    Structured Review

    Millipore mutant u3 snorna genes
    Fluorescein-labeled <t>U3</t> snoRNA is functional in rRNA processing. rRNA precursors (40, 32, and 20S) and products (28 and 18S) were labeled in vivo with [ 32 P]UTP, and the isolated nuclear RNA was subjected to agarose gel electrophoresis. 18S rRNA is not formed after antisense oligonucleotide depletion of endogenous U3 snoRNA but is formed after subsequent injection of U3 snoRNA (0.3 and 1.0 μg/μl) with or without fluorescein (FL) label to rescue rRNA processing.
    Mutant U3 Snorna Genes, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 13510 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nucleolar Localization Elements of Xenopus laevis U3 Small Nucleolar RNA"

    Article Title: Nucleolar Localization Elements of Xenopus laevis U3 Small Nucleolar RNA

    Journal: Molecular Biology of the Cell

    doi:

    Fluorescein-labeled U3 snoRNA is functional in rRNA processing. rRNA precursors (40, 32, and 20S) and products (28 and 18S) were labeled in vivo with [ 32 P]UTP, and the isolated nuclear RNA was subjected to agarose gel electrophoresis. 18S rRNA is not formed after antisense oligonucleotide depletion of endogenous U3 snoRNA but is formed after subsequent injection of U3 snoRNA (0.3 and 1.0 μg/μl) with or without fluorescein (FL) label to rescue rRNA processing.
    Figure Legend Snippet: Fluorescein-labeled U3 snoRNA is functional in rRNA processing. rRNA precursors (40, 32, and 20S) and products (28 and 18S) were labeled in vivo with [ 32 P]UTP, and the isolated nuclear RNA was subjected to agarose gel electrophoresis. 18S rRNA is not formed after antisense oligonucleotide depletion of endogenous U3 snoRNA but is formed after subsequent injection of U3 snoRNA (0.3 and 1.0 μg/μl) with or without fluorescein (FL) label to rescue rRNA processing.

    Techniques Used: Labeling, Functional Assay, In Vivo, Isolation, Agarose Gel Electrophoresis, Injection

    Evolutionarily Conserved Boxes C and D Are Necessary for U3 snoRNA Localization to Nucleoli
    Figure Legend Snippet: Evolutionarily Conserved Boxes C and D Are Necessary for U3 snoRNA Localization to Nucleoli

    Techniques Used:

    Mutant U3 snoRNAs are stable 2 h after injection. 32 P-labeled U3 snoRNA (mutants or wild type) were injected into oocyte nuclei, and the isolated RNA was analyzed by gel electrophoresis 2 h after injection. 32 P-labeled U2 snRNA was coinjected as an internal control to normalize for any differences in injection or recovery of the samples. The stability results are from different experiments for the various mutants, and the amounts of coinjected U3 vs. U2 varied between experiments. For any given mutation, the ratio of U3 to U2 between 0 and 2 h shows that all the mutants are stable at the 2-h time point used for analysis of nucleolar localization, except for the Box C′ mutant, which has partially degraded relative to the U2 snRNA internal control.
    Figure Legend Snippet: Mutant U3 snoRNAs are stable 2 h after injection. 32 P-labeled U3 snoRNA (mutants or wild type) were injected into oocyte nuclei, and the isolated RNA was analyzed by gel electrophoresis 2 h after injection. 32 P-labeled U2 snRNA was coinjected as an internal control to normalize for any differences in injection or recovery of the samples. The stability results are from different experiments for the various mutants, and the amounts of coinjected U3 vs. U2 varied between experiments. For any given mutation, the ratio of U3 to U2 between 0 and 2 h shows that all the mutants are stable at the 2-h time point used for analysis of nucleolar localization, except for the Box C′ mutant, which has partially degraded relative to the U2 snRNA internal control.

    Techniques Used: Mutagenesis, Injection, Labeling, Isolation, Nucleic Acid Electrophoresis

    U3 wild-type snoRNA localizes to nucleoli regardless of the presence or absence of a cap. Fluorescein-coupled wild-type U3 small nucleolar RNA (WT U3) or the spliceosomal U2 small nuclear RNA (WT U2) or unincorporated fluorescein-UTP (FL-UTP) were injected into the nuclei of Xenopus laevis oocytes. After 2 h, nucleoli were prepared for visualization by phase-contrast (PC) or fluorescence microscopy. The nucleolar rDNA is stained blue by DAPI. In contrast to the control of U2 snRNA, fluorescein-labeled U3 snoRNA (FL, green) with a G-cap or A-cap or without a cap localizes to nucleoli. A lampbrush chromosome is visible in the bottom left of the phase-contrast and DAPI images of injected U2 snRNA. Bar, 10 μm.
    Figure Legend Snippet: U3 wild-type snoRNA localizes to nucleoli regardless of the presence or absence of a cap. Fluorescein-coupled wild-type U3 small nucleolar RNA (WT U3) or the spliceosomal U2 small nuclear RNA (WT U2) or unincorporated fluorescein-UTP (FL-UTP) were injected into the nuclei of Xenopus laevis oocytes. After 2 h, nucleoli were prepared for visualization by phase-contrast (PC) or fluorescence microscopy. The nucleolar rDNA is stained blue by DAPI. In contrast to the control of U2 snRNA, fluorescein-labeled U3 snoRNA (FL, green) with a G-cap or A-cap or without a cap localizes to nucleoli. A lampbrush chromosome is visible in the bottom left of the phase-contrast and DAPI images of injected U2 snRNA. Bar, 10 μm.

    Techniques Used: Injection, Fluorescence, Microscopy, Staining, Labeling

    2) Product Images from "A Polymorphic Multigene Family Encoding an Immunodominant Protein from Babesia microti"

    Article Title: A Polymorphic Multigene Family Encoding an Immunodominant Protein from Babesia microti

    Journal: Journal of Clinical Microbiology

    doi:

    (A) Immunoblot showing mini-induction of clone bmn1-7 in the pET17b vector screened with the positive patient serum pool. T0 the culture lysate with an A 560 of 0.5 at time zero; T3, the culture lysate 3 h after the addition of IPTG. (B) Immunoblot showing reactivity of infected patient serum pool or hamster serum with increasing amounts (5, 10, and 20 μl) of crude B. microti lysate. λ, microliters. Numbers on the left are in base pairs.
    Figure Legend Snippet: (A) Immunoblot showing mini-induction of clone bmn1-7 in the pET17b vector screened with the positive patient serum pool. T0 the culture lysate with an A 560 of 0.5 at time zero; T3, the culture lysate 3 h after the addition of IPTG. (B) Immunoblot showing reactivity of infected patient serum pool or hamster serum with increasing amounts (5, 10, and 20 μl) of crude B. microti lysate. λ, microliters. Numbers on the left are in base pairs.

    Techniques Used: Plasmid Preparation, Infection

    3) Product Images from "A Simplified Protocol for High-Yield Expression and Purification of Bacterial Topoisomerase I"

    Article Title: A Simplified Protocol for High-Yield Expression and Purification of Bacterial Topoisomerase I

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2016.04.010

    SDS-PAGE gels of Sm TopI_16b purification results. L, ladder; CE, clarified extract from auto-induction growth; FT, nickel column flow-through; P1, nickel column first peak from first step elution using high salt buffers; Ni 2+ , target fraction from nickel
    Figure Legend Snippet: SDS-PAGE gels of Sm TopI_16b purification results. L, ladder; CE, clarified extract from auto-induction growth; FT, nickel column flow-through; P1, nickel column first peak from first step elution using high salt buffers; Ni 2+ , target fraction from nickel

    Techniques Used: SDS Page, Purification, Nickel Column, Flow Cytometry

    Chromatogram of nickel column linear elution from preliminary construct trials. (A) N-term hexa-His-tagged SmTopI_15b construct. (B) N-term deca-His-tagged SmTopI_16b construct. (C) C-term hexa-His-tagged SmTopI_21d construct. (D) N-term SUMO and hexa-his-tagged
    Figure Legend Snippet: Chromatogram of nickel column linear elution from preliminary construct trials. (A) N-term hexa-His-tagged SmTopI_15b construct. (B) N-term deca-His-tagged SmTopI_16b construct. (C) C-term hexa-His-tagged SmTopI_21d construct. (D) N-term SUMO and hexa-his-tagged

    Techniques Used: Nickel Column, Construct

    4) Product Images from "A Simplified Protocol for High-Yield Expression and Purification of Bacterial Topoisomerase I"

    Article Title: A Simplified Protocol for High-Yield Expression and Purification of Bacterial Topoisomerase I

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2016.04.010

    Chromatogram of nickel column linear elution from preliminary construct trials. (A) N-term hexa-His-tagged SmTopI_15b construct. (B) N-term deca-His-tagged SmTopI_16b construct. (C) C-term hexa-His-tagged SmTopI_21d construct. (D) N-term SUMO and hexa-his-tagged
    Figure Legend Snippet: Chromatogram of nickel column linear elution from preliminary construct trials. (A) N-term hexa-His-tagged SmTopI_15b construct. (B) N-term deca-His-tagged SmTopI_16b construct. (C) C-term hexa-His-tagged SmTopI_21d construct. (D) N-term SUMO and hexa-his-tagged

    Techniques Used: Nickel Column, Construct

    5) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.
    Figure Legend Snippet: MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.

    Techniques Used: Expressing, Staining, Fluorescence

    6) Product Images from "PAPTi: A Peptide Aptamer Interference Toolkit for Perturbation of Protein-Protein Interaction Networks"

    Article Title: PAPTi: A Peptide Aptamer Interference Toolkit for Perturbation of Protein-Protein Interaction Networks

    Journal: Scientific Reports

    doi: 10.1038/srep01156

    Schematic representations of PAPTi platform. (A) PPI network functional analysis. (B) FN3 monobody scaffold. Structure of the FN 3 Monobo dy scaffold 9 13 (FNDY, left) used in this study. The scaffold has an IgG fold similar to the antibody IgG (right). The randomized loops in this study (BC loop and FG loop) within the FNDY scaffold are colored in yellow. As a comparison, the three antigen recognition variable regions within the antibody IgG scaffold are colored in red. (C) PAPTi flowchart: (1) Target protein expression: Target proteins are divided into folded domains and each individual domain is expressed, purified and biotinylated. (2) Phage library construction: Diversified FN3 domain monobody peptide aptamers (FNDY) are cloned in fusion with M13 phage pIII coat protein, resulting in a phage display library with 2 × 10 10 complexity. (3) Selection of target binding phages. (4) ELISA validation: Individual phage clones are amplified and validated for target binding in 96-well ELISA plates. Positive clones are selected for further characterization. (5) Positive individual FNDY clones can be subcloned into various destination vectors for subsequent applications. (6) Cell-based reporter assays: To select functional peptide aptamers, candidate FNDYs are transfected and expressed in reporter cells, and tested using luciferase-based reporter assays. (7) In vivo functional validation: FNDYs are selected to generate transgenic animals.
    Figure Legend Snippet: Schematic representations of PAPTi platform. (A) PPI network functional analysis. (B) FN3 monobody scaffold. Structure of the FN 3 Monobo dy scaffold 9 13 (FNDY, left) used in this study. The scaffold has an IgG fold similar to the antibody IgG (right). The randomized loops in this study (BC loop and FG loop) within the FNDY scaffold are colored in yellow. As a comparison, the three antigen recognition variable regions within the antibody IgG scaffold are colored in red. (C) PAPTi flowchart: (1) Target protein expression: Target proteins are divided into folded domains and each individual domain is expressed, purified and biotinylated. (2) Phage library construction: Diversified FN3 domain monobody peptide aptamers (FNDY) are cloned in fusion with M13 phage pIII coat protein, resulting in a phage display library with 2 × 10 10 complexity. (3) Selection of target binding phages. (4) ELISA validation: Individual phage clones are amplified and validated for target binding in 96-well ELISA plates. Positive clones are selected for further characterization. (5) Positive individual FNDY clones can be subcloned into various destination vectors for subsequent applications. (6) Cell-based reporter assays: To select functional peptide aptamers, candidate FNDYs are transfected and expressed in reporter cells, and tested using luciferase-based reporter assays. (7) In vivo functional validation: FNDYs are selected to generate transgenic animals.

    Techniques Used: Functional Assay, Expressing, Purification, Clone Assay, Selection, Binding Assay, Enzyme-linked Immunosorbent Assay, Amplification, Transfection, Luciferase, In Vivo, Transgenic Assay

    7) Product Images from "Identification of key residues involved in Si transport by the aquaglyceroporins"

    Article Title: Identification of key residues involved in Si transport by the aquaglyceroporins

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201611598

    Transport characteristics and membrane expression of AQP1 F56G/H180G and AQP10 G62F/G202H . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among eight to nine experiments. The asterisk is used to indicate that the mean is significantly different statistically (*, P
    Figure Legend Snippet: Transport characteristics and membrane expression of AQP1 F56G/H180G and AQP10 G62F/G202H . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among eight to nine experiments. The asterisk is used to indicate that the mean is significantly different statistically (*, P

    Techniques Used: Expressing

    Illustrative experiments. (A) Water transport. After incubation in plain water, an oocyte expressing AQP1 and an oocyte expressing AQP10 were microphotographed at different time points for cell volume measurements. (B) Expression of wild-type and mutant AQPs by Western blot analyses. Samples correspond to cell surface proteins detected with an anti-AQP1 Ab (left) or an anti-AQP10 Ab (right). In each panel, water-injected oocytes were used as negative controls. (C) Immunofluorescence experiments. Cryosections postfixed in paraformaldehyde were obtained from AQP-expressing oocytes after a 3-d incubation in medium B1 at 18°C. Wild-type and mutant AQPs were detected with an anti-AQP1 Ab (left) or an anti-AQP10 Ab (right). For each Ab, water-injected oocytes were used as negative controls. Micrographs were taken under confocal microscopy and are shown in the panel for some of the AQPs: (top left) water, (second left) AQP1 1-10-1 , (third left) AQP1 LGRND→IFATY , (bottom left) AQP1 L84C , (top right) water, (second right) wild-type AQP10, (third right) AQP10 N208Y , and (bottom right) AQP10 G62F/G202H . Note that each of the micrographs is ~300 μm in actual width.
    Figure Legend Snippet: Illustrative experiments. (A) Water transport. After incubation in plain water, an oocyte expressing AQP1 and an oocyte expressing AQP10 were microphotographed at different time points for cell volume measurements. (B) Expression of wild-type and mutant AQPs by Western blot analyses. Samples correspond to cell surface proteins detected with an anti-AQP1 Ab (left) or an anti-AQP10 Ab (right). In each panel, water-injected oocytes were used as negative controls. (C) Immunofluorescence experiments. Cryosections postfixed in paraformaldehyde were obtained from AQP-expressing oocytes after a 3-d incubation in medium B1 at 18°C. Wild-type and mutant AQPs were detected with an anti-AQP1 Ab (left) or an anti-AQP10 Ab (right). For each Ab, water-injected oocytes were used as negative controls. Micrographs were taken under confocal microscopy and are shown in the panel for some of the AQPs: (top left) water, (second left) AQP1 1-10-1 , (third left) AQP1 LGRND→IFATY , (bottom left) AQP1 L84C , (top right) water, (second right) wild-type AQP10, (third right) AQP10 N208Y , and (bottom right) AQP10 G62F/G202H . Note that each of the micrographs is ~300 μm in actual width.

    Techniques Used: Incubation, Expressing, Mutagenesis, Western Blot, Injection, Immunofluorescence, Confocal Microscopy

    Location of residues that were substituted in AQP1 and AQP10. The cartons were drawn as described in Fig. 1 using the same color code. (A and B) Chimeras. (C and D) Point substitutions.
    Figure Legend Snippet: Location of residues that were substituted in AQP1 and AQP10. The cartons were drawn as described in Fig. 1 using the same color code. (A and B) Chimeras. (C and D) Point substitutions.

    Techniques Used:

    Transport characteristics and membrane expression of AQP1 Y186N and AQP10 N208Y . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three to four experiments. The asterisk is used to indicate that the mean is significantly different statistically (*, P
    Figure Legend Snippet: Transport characteristics and membrane expression of AQP1 Y186N and AQP10 N208Y . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three to four experiments. The asterisk is used to indicate that the mean is significantly different statistically (*, P

    Techniques Used: Expressing

    Transport characteristics and membrane expression of AQP1 LGRND→IFATY and AQP10 IFATY→LGRND . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three experiments. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (A and B) The asterisk is used to indicate that the mean is significantly different statistically (*, P
    Figure Legend Snippet: Transport characteristics and membrane expression of AQP1 LGRND→IFATY and AQP10 IFATY→LGRND . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three experiments. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (A and B) The asterisk is used to indicate that the mean is significantly different statistically (*, P

    Techniques Used: Expressing, Permeability

    Si influx in oocytes expressing AQP1, expressing AQP10, or injected with water. (A) Oocytes incubated in medium B2 for 90 min at room temperature were assayed for Si content. Data are expressed as means ± SE of 10 oocytes among 25–26 experiments, using the asterisk to indicate that they are significantly different statistically (*, P
    Figure Legend Snippet: Si influx in oocytes expressing AQP1, expressing AQP10, or injected with water. (A) Oocytes incubated in medium B2 for 90 min at room temperature were assayed for Si content. Data are expressed as means ± SE of 10 oocytes among 25–26 experiments, using the asterisk to indicate that they are significantly different statistically (*, P

    Techniques Used: Expressing, Injection, Incubation

    Transport characteristics and membrane expression of AQP1 1-10-1 and AQP10 10-1-10 . (A) Si influx. Oocytes incubated in medium B2 for 90 min at room temperature were assayed for Si content. They were from 10 oocytes among three to four experiments. According to this presentation, a mean of 1 indicates no n -fold differences (Δ) in Si influx. (B) Water permeability. Oocytes incubated in plain water were assayed for volume measurements as described in Materials and methods. They were from three to five oocytes among three experiments. (A and B) Data are expressed as n -fold differences ± SE between AQP1 1-10-1 and AQP1 (left bar) or between AQP10 10-1-10 and AQP10 (right bar) after background subtraction and normalization to channel expression levels. (C) AQP expression at the cell surface ( EXPcs ). Oocytes incubated in medium B2 for 90 min were lysed for Western blot analyses using specific anti-AQP Abs, and the signals obtained were quantified through densitometry. Data are expressed as n -fold differences ± SE between AQP1 1-10-1 and AQP1 (left bar) or between AQP10 10-1-10 and AQP10 (right bar) after background subtraction. They were from three experiments. (A–C) The asterisk is used to indicate that the mean is significantly different statistically (*, P
    Figure Legend Snippet: Transport characteristics and membrane expression of AQP1 1-10-1 and AQP10 10-1-10 . (A) Si influx. Oocytes incubated in medium B2 for 90 min at room temperature were assayed for Si content. They were from 10 oocytes among three to four experiments. According to this presentation, a mean of 1 indicates no n -fold differences (Δ) in Si influx. (B) Water permeability. Oocytes incubated in plain water were assayed for volume measurements as described in Materials and methods. They were from three to five oocytes among three experiments. (A and B) Data are expressed as n -fold differences ± SE between AQP1 1-10-1 and AQP1 (left bar) or between AQP10 10-1-10 and AQP10 (right bar) after background subtraction and normalization to channel expression levels. (C) AQP expression at the cell surface ( EXPcs ). Oocytes incubated in medium B2 for 90 min were lysed for Western blot analyses using specific anti-AQP Abs, and the signals obtained were quantified through densitometry. Data are expressed as n -fold differences ± SE between AQP1 1-10-1 and AQP1 (left bar) or between AQP10 10-1-10 and AQP10 (right bar) after background subtraction. They were from three experiments. (A–C) The asterisk is used to indicate that the mean is significantly different statistically (*, P

    Techniques Used: Expressing, Incubation, Permeability, Western Blot

    Hydropathy plot models and transport characteristics of wild-type AQP1 and AQP10. (A) Model of AQP1. Each symbol corresponds to a single residue within a transmembrane domain (red), a connecting segment (pink), the XX/R filter (yellow), or the NPA motifs (black). Residues above the transmembrane domains face the extracellular side of the membrane. The cartoon was drawn with the program PLOT based on the hydropathy model of Murata et al. (2000) . (B) Model of AQP10. Each symbol corresponds to a single residue in a transmembrane domain (blue), a connecting segment (pale blue), the XX/R filter (brown), or the NPA motifs (black). The cartoon was drawn with the program PLOT based on sequence alignments of transmembrane domains with AQP1. (C) Multiple alignment analysis of AQP family members with Clustal Omega. The residue segment used for each of the channels corresponds to the one that is flanked by green lines in A and B; the N and C termini were excluded from the analysis given that they are poorly conserved among the isoforms. AQP11 and AQP12 were also excluded given that they share much lower homologies with the other family members. Gray boxes correspond to transmembrane segments based on alignments with AQP1, and colored residues correspond to those that were interchanged between AQP1 and AQP10 in this study.
    Figure Legend Snippet: Hydropathy plot models and transport characteristics of wild-type AQP1 and AQP10. (A) Model of AQP1. Each symbol corresponds to a single residue within a transmembrane domain (red), a connecting segment (pink), the XX/R filter (yellow), or the NPA motifs (black). Residues above the transmembrane domains face the extracellular side of the membrane. The cartoon was drawn with the program PLOT based on the hydropathy model of Murata et al. (2000) . (B) Model of AQP10. Each symbol corresponds to a single residue in a transmembrane domain (blue), a connecting segment (pale blue), the XX/R filter (brown), or the NPA motifs (black). The cartoon was drawn with the program PLOT based on sequence alignments of transmembrane domains with AQP1. (C) Multiple alignment analysis of AQP family members with Clustal Omega. The residue segment used for each of the channels corresponds to the one that is flanked by green lines in A and B; the N and C termini were excluded from the analysis given that they are poorly conserved among the isoforms. AQP11 and AQP12 were also excluded given that they share much lower homologies with the other family members. Gray boxes correspond to transmembrane segments based on alignments with AQP1, and colored residues correspond to those that were interchanged between AQP1 and AQP10 in this study.

    Techniques Used: Sequencing

    Transport characteristics and membrane expression of AQP1 L84C and AQP10 C90L . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three to six experiments. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (B) The asterisk is used to indicate that the mean is significantly different statistically (*, P
    Figure Legend Snippet: Transport characteristics and membrane expression of AQP1 L84C and AQP10 C90L . Experimental conditions were as described in Fig. 4 . Data expression is also as described in Fig. 4 . (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three to six experiments. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (B) The asterisk is used to indicate that the mean is significantly different statistically (*, P

    Techniques Used: Expressing, Permeability

    8) Product Images from "Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy"

    Article Title: Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-0978-6

    Effects of different chaperones on HPV His 6 -E6 expression levels. Representative immunoblots of induction and purification of HPV16 His 6 -E6 produced in E. coli JM109. a Bacterial transformation with the pQE30-HPV16 His 6 -E6 expression plasmid alone (w/o) or with the different chaperone systems (chaperones A–E, see Table 2 ). Total proteins were extracted by re-suspension in SDS-loading buffer of E. coli cell cultures, according to their OD 600 . b Purification of the HPV16 His 6 -E6 protein without chaperones (w/o) and with the trigger factor chaperone (with chaperones; chaperone E, see Table 2 ). M ColorBurst, Sigma, lanes 1 1 µL, lanes 2 3 µL, lane 3 C + , 20 ng of purified His 6 -E6 protein positive control
    Figure Legend Snippet: Effects of different chaperones on HPV His 6 -E6 expression levels. Representative immunoblots of induction and purification of HPV16 His 6 -E6 produced in E. coli JM109. a Bacterial transformation with the pQE30-HPV16 His 6 -E6 expression plasmid alone (w/o) or with the different chaperone systems (chaperones A–E, see Table 2 ). Total proteins were extracted by re-suspension in SDS-loading buffer of E. coli cell cultures, according to their OD 600 . b Purification of the HPV16 His 6 -E6 protein without chaperones (w/o) and with the trigger factor chaperone (with chaperones; chaperone E, see Table 2 ). M ColorBurst, Sigma, lanes 1 1 µL, lanes 2 3 µL, lane 3 C + , 20 ng of purified His 6 -E6 protein positive control

    Techniques Used: Expressing, Western Blot, Purification, Produced, Electroporation Bacterial Transformation, Plasmid Preparation, Positive Control

    9) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.
    Figure Legend Snippet: MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.

    Techniques Used: Expressing, Staining, Fluorescence

    10) Product Images from "Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos"

    Article Title: Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2010.11

    Chromosome instability and centrosome amplification in p53 −/− tetraploid HCT 116 clones. ( A , B ) p53 deficiency increases the percentage of unstable tetraploid clones. Tetraploid HCT 116 clones were generated from wild type (WT), p53 −/− , Bax −/− , p21 −/− . Cell cycle distribution and apoptosis-related parameters were evaluated 4 weeks after cloning by multiparametric cytofluorometry upon staining with Hoechst 33342 (which measures DNA content), the mitochondrial transmembrane potential (ΔΨ m )-sensitive dye 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) and propidium iodide (PI, an exclusion dye that only stains dead cells). Representative plots are shown in A , the inserts therein showing positive controls for ΔΨ m dissipation (as obtained by treating the cells for 30 min with 100 μM protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP) and plasma membrane permeabilization (as resulting from a 2 min-long incubation in 0.5% (w/v) saponin (Sapo)). Tetraploid clones were classified according to genomic instability (as evaluated by the quantification of viable sub-tetraploid populations) in stable (sub-tetraploid cells
    Figure Legend Snippet: Chromosome instability and centrosome amplification in p53 −/− tetraploid HCT 116 clones. ( A , B ) p53 deficiency increases the percentage of unstable tetraploid clones. Tetraploid HCT 116 clones were generated from wild type (WT), p53 −/− , Bax −/− , p21 −/− . Cell cycle distribution and apoptosis-related parameters were evaluated 4 weeks after cloning by multiparametric cytofluorometry upon staining with Hoechst 33342 (which measures DNA content), the mitochondrial transmembrane potential (ΔΨ m )-sensitive dye 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) and propidium iodide (PI, an exclusion dye that only stains dead cells). Representative plots are shown in A , the inserts therein showing positive controls for ΔΨ m dissipation (as obtained by treating the cells for 30 min with 100 μM protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP) and plasma membrane permeabilization (as resulting from a 2 min-long incubation in 0.5% (w/v) saponin (Sapo)). Tetraploid clones were classified according to genomic instability (as evaluated by the quantification of viable sub-tetraploid populations) in stable (sub-tetraploid cells

    Techniques Used: Amplification, Clone Assay, Generated, Staining, Incubation

    Phase 2 unstable p53 −/− tetraploid clones form aggressive tumours. ( A ) Nude mice were subcutaneously xenografted with similar numbers of wild type (WT) or tetraploid cells that were fluorescence-activated cell sorter (FACS)-purified from p53 −/− tetraploid clones of the indicated type (stable, phase 1 or 2 unstable). Tumour volumes (mean±s.e.m., n =10 mice for each clone type) were monitored over time. ( B , C ) Tumour cells were recovered from mice ( in vivo ) and the DNA-content was assessed by propidium iodide (PI) staining and cytofluorometry. The same was done on aliquots of the FACS-purified parental cells that were kept frozen or that were cultured in vitro for the same time as tumours proliferated in vivo . B reports representative cell cycle distributions of tumour cells of the indicated type upon recovery from mice. Percentages refer to the sub-tetraploid populations. C depicts the percentage of sub-tetraploid cells (mean±s.e.m., n =3 independent assessments) observed for the indicated cell type.
    Figure Legend Snippet: Phase 2 unstable p53 −/− tetraploid clones form aggressive tumours. ( A ) Nude mice were subcutaneously xenografted with similar numbers of wild type (WT) or tetraploid cells that were fluorescence-activated cell sorter (FACS)-purified from p53 −/− tetraploid clones of the indicated type (stable, phase 1 or 2 unstable). Tumour volumes (mean±s.e.m., n =10 mice for each clone type) were monitored over time. ( B , C ) Tumour cells were recovered from mice ( in vivo ) and the DNA-content was assessed by propidium iodide (PI) staining and cytofluorometry. The same was done on aliquots of the FACS-purified parental cells that were kept frozen or that were cultured in vitro for the same time as tumours proliferated in vivo . B reports representative cell cycle distributions of tumour cells of the indicated type upon recovery from mice. Percentages refer to the sub-tetraploid populations. C depicts the percentage of sub-tetraploid cells (mean±s.e.m., n =3 independent assessments) observed for the indicated cell type.

    Techniques Used: Clone Assay, Mouse Assay, Fluorescence, FACS, Purification, In Vivo, Staining, Cell Culture, In Vitro

    Characterization and fate of the sub-tetraploid offspring of p53 −/− tetraploid cells. ( A – C ) An important fraction of sub-tetraploid cells are aneuploid. Sub-tetraploid cells derived from phase 1 and 2 unstable p53 −/− tetraploid HCT 116 clones or the 2n population of the parental p53 −/− diploid cell line were fluorescence-activated cell sorter (FACS)-purified as indicated in A . Thereafter, nuclei from the sorted populations were subjected to fluorescence in situ hybridization (FISH) with probes specific for the centromeric region of chromosome 8, 10 and 18 (green, red and blue fluorescence, respectively). Representative immunofluorescence microphotographs are shown in B . In C the percentage of wild type (WT) diploid or p53 −/− sub-tetraploid cells characterized by the indicated chromosomal setup is reported, as scored among ⩾100 metaphases for each cell type (mean±s.e.m., n =3 independent determinations). Alternatively, FACS-purified cells obtained as in A were subjected to clonogenic survival assays ( D ). Columns depict the survival fraction (mean±s.e.m., n =3 independent experiments carried out in triplicate with at least three distinct clones for each type) of WT diploid or sub-tetraploid cells derived from p53 −/− tetraploid clones of the indicated class. ( E , F ) Videomicroscopy of sub-tetraploid cells. p53 −/− diploid cells and tetraploid HCT 116 clones expressing a histone 2B–green fluorescent protein chimera (H2B–GFP) were subjected to FACS-purification of diploid or sub-tetraploid populations as in A . F provides a graphic representation and quantitative data (%, mean±s.e.m., n =3 independent experiment, 100-150 cells analyzed) about the fate of WT diploid or p53 −/− .
    Figure Legend Snippet: Characterization and fate of the sub-tetraploid offspring of p53 −/− tetraploid cells. ( A – C ) An important fraction of sub-tetraploid cells are aneuploid. Sub-tetraploid cells derived from phase 1 and 2 unstable p53 −/− tetraploid HCT 116 clones or the 2n population of the parental p53 −/− diploid cell line were fluorescence-activated cell sorter (FACS)-purified as indicated in A . Thereafter, nuclei from the sorted populations were subjected to fluorescence in situ hybridization (FISH) with probes specific for the centromeric region of chromosome 8, 10 and 18 (green, red and blue fluorescence, respectively). Representative immunofluorescence microphotographs are shown in B . In C the percentage of wild type (WT) diploid or p53 −/− sub-tetraploid cells characterized by the indicated chromosomal setup is reported, as scored among ⩾100 metaphases for each cell type (mean±s.e.m., n =3 independent determinations). Alternatively, FACS-purified cells obtained as in A were subjected to clonogenic survival assays ( D ). Columns depict the survival fraction (mean±s.e.m., n =3 independent experiments carried out in triplicate with at least three distinct clones for each type) of WT diploid or sub-tetraploid cells derived from p53 −/− tetraploid clones of the indicated class. ( E , F ) Videomicroscopy of sub-tetraploid cells. p53 −/− diploid cells and tetraploid HCT 116 clones expressing a histone 2B–green fluorescent protein chimera (H2B–GFP) were subjected to FACS-purification of diploid or sub-tetraploid populations as in A . F provides a graphic representation and quantitative data (%, mean±s.e.m., n =3 independent experiment, 100-150 cells analyzed) about the fate of WT diploid or p53 −/− .

    Techniques Used: Derivative Assay, Clone Assay, Fluorescence, FACS, Purification, In Situ Hybridization, Fluorescence In Situ Hybridization, Immunofluorescence, Expressing

    Correlation between Mos expression and genomic instability in p53 −/− tetraploid clones. ( A ) Mos is upregulated in unstable tetraploid clones. Protein extracts obtained from wild type (WT) or p53 −/− clones of the indicated type (S=stable, P1=phase 1, P2=phase 2), were subjected to immunoblotting with antibodies that specifically recognize Mos or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, monitored as a loading control). ( B ) Mos overexpression triggers genomic instability in stable tetraploid cells. Stable p53 −/− HCT 116 cells were transfected for 48 h with a polycystronic plasmid expressing green fluorescent protein (GFP) alone or co-expressing Mos and GFP. The GFP + populations were fluorescence-activated cell sorter (FACS)-purified, cultured for 48 h, and then subjected to subsequent rounds of transfections with the same constructs at intervals of ∼5 days. Columns depict the percentage of sub-tetraploid cells (mean±s.e.m., n . ( C ) Mos depletion stabilizes unstable p53 −/− tetraploid clones. Phase 1 unstable p53 −/− . The percentage of sub-tetraploid cells (mean±s.e.m., n =5 independent experiments) was determined. The efficacy of the small interfering RNA (siRNA)-mediated downregulation of Mos was confirmed by immunoblotting (insert). ( D ) A non-interferable Mos variant restores genomic instability of originally unstable p53 −/− tetraploid cells stabilized by transfection with siRNAs that deplete endogenous Mos. Phase 1 unstable p53 −/− tetraploid HCT 116 cells were transfected with a control (UNR) or a Mos-depleting siRNA (Mos_1) for 24 h, followed by further transfection with a control vector encoding green fluorescent protein (GFP) alone or with constructs for the co-expression of GFP and wild type (Mos–GFP) or mutant non-interferable Mos (Mos mut –GFP). After 48 h, Mos expression was controlled by immunoblotting (insert), and 24 h later the percentage of sub-tetraploid cells was quantified (mean±s.e.m., n =3 independent assessments). ( E ) Mos depletion avoids genomic instability caused by p53 knockdown. Wild type (WT) tetraploid HCT 116 clones were repeatedly (6 times) transfected with a control (UNR), a p53-specific (p53_2) and a Mos-specific (Mos_1) siRNA alone or in combination for 72 h, followed by DNA content analysis and quantification of sub-tetraploid cells. The histogram summarizes data (mean±s.e.m.) from five independent experiments.
    Figure Legend Snippet: Correlation between Mos expression and genomic instability in p53 −/− tetraploid clones. ( A ) Mos is upregulated in unstable tetraploid clones. Protein extracts obtained from wild type (WT) or p53 −/− clones of the indicated type (S=stable, P1=phase 1, P2=phase 2), were subjected to immunoblotting with antibodies that specifically recognize Mos or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, monitored as a loading control). ( B ) Mos overexpression triggers genomic instability in stable tetraploid cells. Stable p53 −/− HCT 116 cells were transfected for 48 h with a polycystronic plasmid expressing green fluorescent protein (GFP) alone or co-expressing Mos and GFP. The GFP + populations were fluorescence-activated cell sorter (FACS)-purified, cultured for 48 h, and then subjected to subsequent rounds of transfections with the same constructs at intervals of ∼5 days. Columns depict the percentage of sub-tetraploid cells (mean±s.e.m., n . ( C ) Mos depletion stabilizes unstable p53 −/− tetraploid clones. Phase 1 unstable p53 −/− . The percentage of sub-tetraploid cells (mean±s.e.m., n =5 independent experiments) was determined. The efficacy of the small interfering RNA (siRNA)-mediated downregulation of Mos was confirmed by immunoblotting (insert). ( D ) A non-interferable Mos variant restores genomic instability of originally unstable p53 −/− tetraploid cells stabilized by transfection with siRNAs that deplete endogenous Mos. Phase 1 unstable p53 −/− tetraploid HCT 116 cells were transfected with a control (UNR) or a Mos-depleting siRNA (Mos_1) for 24 h, followed by further transfection with a control vector encoding green fluorescent protein (GFP) alone or with constructs for the co-expression of GFP and wild type (Mos–GFP) or mutant non-interferable Mos (Mos mut –GFP). After 48 h, Mos expression was controlled by immunoblotting (insert), and 24 h later the percentage of sub-tetraploid cells was quantified (mean±s.e.m., n =3 independent assessments). ( E ) Mos depletion avoids genomic instability caused by p53 knockdown. Wild type (WT) tetraploid HCT 116 clones were repeatedly (6 times) transfected with a control (UNR), a p53-specific (p53_2) and a Mos-specific (Mos_1) siRNA alone or in combination for 72 h, followed by DNA content analysis and quantification of sub-tetraploid cells. The histogram summarizes data (mean±s.e.m.) from five independent experiments.

    Techniques Used: Expressing, Clone Assay, Over Expression, Transfection, Plasmid Preparation, Fluorescence, FACS, Purification, Cell Culture, Construct, Small Interfering RNA, Variant Assay, Mutagenesis

    Effect of p53 on the survival and on the genomic stability of tetraploid HCT 116 cells. ( A , B ) The absence of p53 increases the clonogenic survival of freshly generated polyploid cells. Wild type (WT), p53 −/− , Bax −/− and p21 −/− diploid human colon carcinoma HCT 116 cells and HCT 116 cells stably transfected with a plasmid encoding the baculoviral inhibitor of caspases p35 (p35) were left untreated or treated with 100 nM nocodazole (Noco) for 48 h. After washing, cells were cultured for additional 48 h in drug-free culture medium, then stained with Hoechst 33342, followed by fluorescence-activated cell sorter (FACS) purification of diploid (2n, white and grey symbols for untreated and nocodazole-treated cells, respectively) or polyploid ( > 4n, black symbols) cell populations. In A , representative cell cycle distributions of WT and p53 −/− cells from one out of three independent experiments are shown. X -axis = Hoechst 33342 fluorescence (DNA content); Y -axis = cell number per channel (counts). In B , the results of clonogenic survival assays carried out on such FACS-purified cells are reported. The upper part shows representative pictures of colonies formed by WT and p53 −/− cells as observed upon crystal violet staining 10 days after FACS purification. Columns depict the survival fraction (mean±s.e.m., n =3 parallel wells, normalized for plating efficiency and to control diploid cells) of the diploid and polyploid cell populations with the indicated genotype and corresponding to the FACS-purified populations represented in A . Asterisks indicate statistically significant differences as compared to WT tetraploid cells (Student's t -test, P
    Figure Legend Snippet: Effect of p53 on the survival and on the genomic stability of tetraploid HCT 116 cells. ( A , B ) The absence of p53 increases the clonogenic survival of freshly generated polyploid cells. Wild type (WT), p53 −/− , Bax −/− and p21 −/− diploid human colon carcinoma HCT 116 cells and HCT 116 cells stably transfected with a plasmid encoding the baculoviral inhibitor of caspases p35 (p35) were left untreated or treated with 100 nM nocodazole (Noco) for 48 h. After washing, cells were cultured for additional 48 h in drug-free culture medium, then stained with Hoechst 33342, followed by fluorescence-activated cell sorter (FACS) purification of diploid (2n, white and grey symbols for untreated and nocodazole-treated cells, respectively) or polyploid ( > 4n, black symbols) cell populations. In A , representative cell cycle distributions of WT and p53 −/− cells from one out of three independent experiments are shown. X -axis = Hoechst 33342 fluorescence (DNA content); Y -axis = cell number per channel (counts). In B , the results of clonogenic survival assays carried out on such FACS-purified cells are reported. The upper part shows representative pictures of colonies formed by WT and p53 −/− cells as observed upon crystal violet staining 10 days after FACS purification. Columns depict the survival fraction (mean±s.e.m., n =3 parallel wells, normalized for plating efficiency and to control diploid cells) of the diploid and polyploid cell populations with the indicated genotype and corresponding to the FACS-purified populations represented in A . Asterisks indicate statistically significant differences as compared to WT tetraploid cells (Student's t -test, P

    Techniques Used: Generated, Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Staining, Fluorescence, FACS, Purification

    Effects of Mos on centrosome coalescence in p53 −/− tetraploid clones. ( A ) Mos localizes to centrosomes. Immunofluorescence staining for the detection of β- or γ-tubulin (both in red) in p53 −/− . ( B ) Mos depletion partially reverts genomic instability induced by HSET downregulation. Stable and unstable p53 −/− tetraploid HCT 116 clones were transfected with a control (UNR), a Mos-specific (Mos_1) or a HSET-specific (HSET_1) small interfering RNA (siRNA) alone or in combination for 72 h, followed by cytofluorometric analysis of DNA content and quantification of sub-tetraploid cells (mean±s.e.m., n =3 independent experiments). ( C , D ) Increased coalescence and inactivation of supernumerary centrosomes upon Mos depletion. Unstable p53 −/− tetraploid HCT 116 cells were co-transfected with the indicated siRNA combinations for 72 h, and then subjected to immunofluorescence staining for the detection of β- and γ-tubulin (green and red fluorescence, respectively). Hoechst 33342 was employed for nuclear counterstaining. C shows representative immunofluorescence microphotographs of supernumerary centrosomes coalescing or being inactivated (c i . D depicts the percentage of metaphases (mean±s.e.m., n =3 independent experiments) characterized by the indicated mitotic spindle arrangements, as scored among at least 100 cells per for each condition. All experiments have been performed on at least three distinct clones of each type. ( E , F ) Live-cell imaging of the Mos effects on centrosomal and chromosomal dynamics. A (phase 2) unstable tetraploid p53 −/− GFP–H2B-expressing clone was transiently transfected with a Mos-specific or a control siRNA (UNR) together with a construct coding for centrin–DsRed ( E ). E . Hatched lines indicate the position of daughter cells arising from the observed mitoses. The frequency of multipolar divisions and failed cytokineses observed by live-cell imaging is reported in F (mean±s.e.m., n =3 independent experiments).
    Figure Legend Snippet: Effects of Mos on centrosome coalescence in p53 −/− tetraploid clones. ( A ) Mos localizes to centrosomes. Immunofluorescence staining for the detection of β- or γ-tubulin (both in red) in p53 −/− . ( B ) Mos depletion partially reverts genomic instability induced by HSET downregulation. Stable and unstable p53 −/− tetraploid HCT 116 clones were transfected with a control (UNR), a Mos-specific (Mos_1) or a HSET-specific (HSET_1) small interfering RNA (siRNA) alone or in combination for 72 h, followed by cytofluorometric analysis of DNA content and quantification of sub-tetraploid cells (mean±s.e.m., n =3 independent experiments). ( C , D ) Increased coalescence and inactivation of supernumerary centrosomes upon Mos depletion. Unstable p53 −/− tetraploid HCT 116 cells were co-transfected with the indicated siRNA combinations for 72 h, and then subjected to immunofluorescence staining for the detection of β- and γ-tubulin (green and red fluorescence, respectively). Hoechst 33342 was employed for nuclear counterstaining. C shows representative immunofluorescence microphotographs of supernumerary centrosomes coalescing or being inactivated (c i . D depicts the percentage of metaphases (mean±s.e.m., n =3 independent experiments) characterized by the indicated mitotic spindle arrangements, as scored among at least 100 cells per for each condition. All experiments have been performed on at least three distinct clones of each type. ( E , F ) Live-cell imaging of the Mos effects on centrosomal and chromosomal dynamics. A (phase 2) unstable tetraploid p53 −/− GFP–H2B-expressing clone was transiently transfected with a Mos-specific or a control siRNA (UNR) together with a construct coding for centrin–DsRed ( E ). E . Hatched lines indicate the position of daughter cells arising from the observed mitoses. The frequency of multipolar divisions and failed cytokineses observed by live-cell imaging is reported in F (mean±s.e.m., n =3 independent experiments).

    Techniques Used: Clone Assay, Immunofluorescence, Staining, Transfection, Small Interfering RNA, Fluorescence, Live Cell Imaging, Expressing, Construct

    11) Product Images from "Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases"

    Article Title: Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh995

    Fractionation of P.aerophilum extracts and estimation of the molecular weight of the GO-DNA glycosylase. ( A ) Profile of Fraction V eluted from a HiTrap SP column with a linear gradient from 0.04 to 0.6 M NaCl. Inset shows the GO/G activity in 8 μl of the load (L), flow-through (F), wash (W) and fractions 6–12. Incubations were carried out for 30 min at 60°C, using 1 pmol of the GO/G substrate. The positions of the full-length 60mer substrate and of the 23mer product are indicated. ( B ) Recovery and renaturation of the protein eluted from 11 slices of the 15% SDS–PAGE gel (left panel; M, molecular weight standards; VI, 25 μl of fraction VI). The GO/G-processing activity was found to reside predominantly in slice 9 (right panel).
    Figure Legend Snippet: Fractionation of P.aerophilum extracts and estimation of the molecular weight of the GO-DNA glycosylase. ( A ) Profile of Fraction V eluted from a HiTrap SP column with a linear gradient from 0.04 to 0.6 M NaCl. Inset shows the GO/G activity in 8 μl of the load (L), flow-through (F), wash (W) and fractions 6–12. Incubations were carried out for 30 min at 60°C, using 1 pmol of the GO/G substrate. The positions of the full-length 60mer substrate and of the 23mer product are indicated. ( B ) Recovery and renaturation of the protein eluted from 11 slices of the 15% SDS–PAGE gel (left panel; M, molecular weight standards; VI, 25 μl of fraction VI). The GO/G-processing activity was found to reside predominantly in slice 9 (right panel).

    Techniques Used: Fractionation, Molecular Weight, Activity Assay, Flow Cytometry, SDS Page

    12) Product Images from "Visible Light-Responsive Platinum-Containing Titania Nanoparticle-Mediated Photocatalysis Induces Nucleotide Insertion, Deletion and Substitution Mutations"

    Article Title: Visible Light-Responsive Platinum-Containing Titania Nanoparticle-Mediated Photocatalysis Induces Nucleotide Insertion, Deletion and Substitution Mutations

    Journal: Nanomaterials

    doi: 10.3390/nano7010002

    Detection of mutated clones using  lac Z α-peptide complementation. ( A , B ) After being complemented with  lac Z α-peptide expression, the transformants are displayed as blue colonies on the agar plates with 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside. The VLRP TiO 2 -Pt NP-mediated photocatalysis markedly reduces the number of transformants, compared with the control groups using UV-responsive TiO 2  NPs, under visible light illumination. ( C ) Quantified results show that TiO 2 -Pt photocatalysis can induce the formation of white colonies. This indicates that mutations hit the  lac Zα region because of a loss-of-function (loss-of-complementation) phenotype, compared with the wild-type plasmid-transformed blue colonies. ND: no detected colony. *  p
    Figure Legend Snippet: Detection of mutated clones using lac Z α-peptide complementation. ( A , B ) After being complemented with lac Z α-peptide expression, the transformants are displayed as blue colonies on the agar plates with 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside. The VLRP TiO 2 -Pt NP-mediated photocatalysis markedly reduces the number of transformants, compared with the control groups using UV-responsive TiO 2 NPs, under visible light illumination. ( C ) Quantified results show that TiO 2 -Pt photocatalysis can induce the formation of white colonies. This indicates that mutations hit the lac Zα region because of a loss-of-function (loss-of-complementation) phenotype, compared with the wild-type plasmid-transformed blue colonies. ND: no detected colony. * p

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Transformation Assay

    13) Product Images from "Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases"

    Article Title: Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh995

    Identification of the P.aerophilum GO-glycosylase (PAE2237) by mass spectrometry. ( A ) Left panel: TCA precipitate from 2 ml of Fraction VI (Materials and Methods) was resolved on a 12.5% SDS–PAGE gel and stained with Coomassie blue. M, Bio-Rad SDS–PAGE molecular weight standards. The two bands (1 and 2) detected at ∼30 kDa were cut out and subjected to a tryptic digest (Materials and Methods) followed by MALDI-TOF Mass Spectrometry (MS) analysis of the peptide fragments. Right panel: MALDI-TOF MS analysis of band 1. Measured masses and amino acid sequences of the tryptic peptides are listed in the table. ‘a’ indicates measured minus calculated mass. ( B ) The peptide sequences identified by MS (shown in boldface) could be assigned to ORF PAE2237 in the P.aerophilum genome that encodes a hypothetical protein of 256 amino acids (29.5 kDa).
    Figure Legend Snippet: Identification of the P.aerophilum GO-glycosylase (PAE2237) by mass spectrometry. ( A ) Left panel: TCA precipitate from 2 ml of Fraction VI (Materials and Methods) was resolved on a 12.5% SDS–PAGE gel and stained with Coomassie blue. M, Bio-Rad SDS–PAGE molecular weight standards. The two bands (1 and 2) detected at ∼30 kDa were cut out and subjected to a tryptic digest (Materials and Methods) followed by MALDI-TOF Mass Spectrometry (MS) analysis of the peptide fragments. Right panel: MALDI-TOF MS analysis of band 1. Measured masses and amino acid sequences of the tryptic peptides are listed in the table. ‘a’ indicates measured minus calculated mass. ( B ) The peptide sequences identified by MS (shown in boldface) could be assigned to ORF PAE2237 in the P.aerophilum genome that encodes a hypothetical protein of 256 amino acids (29.5 kDa).

    Techniques Used: Mass Spectrometry, SDS Page, Staining, Molecular Weight

    14) Product Images from "A Simplified Protocol for High-Yield Expression and Purification of Bacterial Topoisomerase I"

    Article Title: A Simplified Protocol for High-Yield Expression and Purification of Bacterial Topoisomerase I

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2016.04.010

    Chromatogram of nickel column linear elution from preliminary construct trials. (A) N-term hexa-His-tagged SmTopI_15b construct. (B) N-term deca-His-tagged SmTopI_16b construct. (C) C-term hexa-His-tagged SmTopI_21d construct. (D) N-term SUMO and hexa-his-tagged
    Figure Legend Snippet: Chromatogram of nickel column linear elution from preliminary construct trials. (A) N-term hexa-His-tagged SmTopI_15b construct. (B) N-term deca-His-tagged SmTopI_16b construct. (C) C-term hexa-His-tagged SmTopI_21d construct. (D) N-term SUMO and hexa-his-tagged

    Techniques Used: Nickel Column, Construct

    15) Product Images from "Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi"

    Article Title: Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    Journal: Microbes and Environments

    doi: 10.1264/jsme2.ME16085

    DGGE patterns of nested PCR products derived from respective parts of wheat, soybean, and potato samples. The products were prepared with the ITS1F KU DNA primer with the GC clamp and ITS2 primer. The symbols “−” and “+” indicate the lanes prepared without and with the LNA technique. The products that sufficiently amplified the fungal ITS regions are used in lane “+”. “C” represents aseptic amplicons to provide the position of the host plant band in the DGGE gel. The sequences of DGGE bands indicated with arrows were obtained in order to identify the closest relatives using the DNA database.
    Figure Legend Snippet: DGGE patterns of nested PCR products derived from respective parts of wheat, soybean, and potato samples. The products were prepared with the ITS1F KU DNA primer with the GC clamp and ITS2 primer. The symbols “−” and “+” indicate the lanes prepared without and with the LNA technique. The products that sufficiently amplified the fungal ITS regions are used in lane “+”. “C” represents aseptic amplicons to provide the position of the host plant band in the DGGE gel. The sequences of DGGE bands indicated with arrows were obtained in order to identify the closest relatives using the DNA database.

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Nested PCR, Derivative Assay, Amplification

    Estimation of effective concentrations for LNA oligonucleotides. LNA oligonucleotides were used in the ranges of 0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, and 4.0 μM. Higher concentrations of 6.0 μM and 8.0 μM were also examined for potato leaf and stem samples. “M” is the marker for 100-bp ladders, and “C” is aseptic amplicons to provide the position of host plant DNA in the agarose gel. “LNA(−)” represents amplicons prepared with the ITS1F DNA primer and ITS4 primer.
    Figure Legend Snippet: Estimation of effective concentrations for LNA oligonucleotides. LNA oligonucleotides were used in the ranges of 0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, and 4.0 μM. Higher concentrations of 6.0 μM and 8.0 μM were also examined for potato leaf and stem samples. “M” is the marker for 100-bp ladders, and “C” is aseptic amplicons to provide the position of host plant DNA in the agarose gel. “LNA(−)” represents amplicons prepared with the ITS1F DNA primer and ITS4 primer.

    Techniques Used: Marker, Agarose Gel Electrophoresis

    16) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.
    Figure Legend Snippet: MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.

    Techniques Used: Expressing, Staining, Fluorescence

    MoSnt2 mediates H3 deacetylation and regulates expression of autophagy genes. (A) Visualization of the interaction between proteins as shown in the BiFC assay. Vegetative hyphae were stained with DAPI and then analyzed by epifluorescence microscopy. Scale bar: 10 μm. (B) GST-PHD1 coimmunoprecipitates H3 histones. E. coli -expressed fusion proteins were used for affinity isolation of histones of calf thymus and immunoblot analysis conducted with the antibodies indicated. The star indicates GST-PHD1 and GST-PHD2, while arrowhead indicates GST. (C) Histone deacetylase activity in affinity isolation complexes. (D) Immunoblot analysis of histone proteins in M. oryzae with the indicated primary antibodies. (E) qRT-PCR analysis on the expression levels of autophagy genes. (F) In vitro affinity isolation of autophagy gene DNA by MoSnt2. GST-MoSnt2-F1, GST-MoSnt2-F2 or GST alone were incubated with sheared chromatin, affinity isolated, washed and subjected to qPCR for autophagy genes. Similar results were obtained from 3 independent biological experiments.
    Figure Legend Snippet: MoSnt2 mediates H3 deacetylation and regulates expression of autophagy genes. (A) Visualization of the interaction between proteins as shown in the BiFC assay. Vegetative hyphae were stained with DAPI and then analyzed by epifluorescence microscopy. Scale bar: 10 μm. (B) GST-PHD1 coimmunoprecipitates H3 histones. E. coli -expressed fusion proteins were used for affinity isolation of histones of calf thymus and immunoblot analysis conducted with the antibodies indicated. The star indicates GST-PHD1 and GST-PHD2, while arrowhead indicates GST. (C) Histone deacetylase activity in affinity isolation complexes. (D) Immunoblot analysis of histone proteins in M. oryzae with the indicated primary antibodies. (E) qRT-PCR analysis on the expression levels of autophagy genes. (F) In vitro affinity isolation of autophagy gene DNA by MoSnt2. GST-MoSnt2-F1, GST-MoSnt2-F2 or GST alone were incubated with sheared chromatin, affinity isolated, washed and subjected to qPCR for autophagy genes. Similar results were obtained from 3 independent biological experiments.

    Techniques Used: Expressing, Bimolecular Fluorescence Complementation Assay, Staining, Epifluorescence Microscopy, Isolation, Histone Deacetylase Assay, Activity Assay, Quantitative RT-PCR, In Vitro, Incubation, Real-time Polymerase Chain Reaction

    Model for MoSnt2-mediated epigenetic control of pathogenicity in M. oryzae . MoTor promotes the expression of MoSNT2 through unidentified effector(s) or transcription factor(s). Nucleus-localized MoSnt2 recognizes acetylated histone H3 and recruits the histone HDAC deacetylase complex to targeted chromatin regions. The MoSnt2-recruited HDAC then deacetylates H3 and alters expression of genes. MoSnt2-regulated gene expression functions to repress autophagy to promote hyphal proliferation during vegetative growth in nutrient rich conditions, while promoting autophagic conidial cell death and assisting pathogenic growth on host rice.
    Figure Legend Snippet: Model for MoSnt2-mediated epigenetic control of pathogenicity in M. oryzae . MoTor promotes the expression of MoSNT2 through unidentified effector(s) or transcription factor(s). Nucleus-localized MoSnt2 recognizes acetylated histone H3 and recruits the histone HDAC deacetylase complex to targeted chromatin regions. The MoSnt2-recruited HDAC then deacetylates H3 and alters expression of genes. MoSnt2-regulated gene expression functions to repress autophagy to promote hyphal proliferation during vegetative growth in nutrient rich conditions, while promoting autophagic conidial cell death and assisting pathogenic growth on host rice.

    Techniques Used: Expressing, Histone Deacetylase Assay

    MoSNT2 is crucial for infection structure development and autophagic cell death. (A) Infection structure development on onion epidermis at 36 hpi. The black arrowhead and arrow indicates conidium and appressorium respectively, while the white arrowhead indicates invasive hypha. (B) Percentage of appressorium and penetration peg formation on onion epidermis (n >  50, triple replications, ** PÂÂ
    Figure Legend Snippet: MoSNT2 is crucial for infection structure development and autophagic cell death. (A) Infection structure development on onion epidermis at 36 hpi. The black arrowhead and arrow indicates conidium and appressorium respectively, while the white arrowhead indicates invasive hypha. (B) Percentage of appressorium and penetration peg formation on onion epidermis (n >  50, triple replications, ** PÂÂ

    Techniques Used: Infection

    MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.
    Figure Legend Snippet: MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, RNA Extraction

    MoSNT2 is critical for growth and reproduction of M. oryzae . (A) Hyphal growth of plate colonies on CM agar medium. (B) Quantified diameters of colonies. Error bars represent standard deviations. Asterisk indicates significant difference (** PÂÂ
    Figure Legend Snippet: MoSNT2 is critical for growth and reproduction of M. oryzae . (A) Hyphal growth of plate colonies on CM agar medium. (B) Quantified diameters of colonies. Error bars represent standard deviations. Asterisk indicates significant difference (** PÂÂ

    Techniques Used:

    MoSNT2 is essential for plant infection by M. oryzae . (A) Rice leaf segments infected with fungal mycelium. (B) Measurement of fungal biomass in infected leaves based on qPCR analysis of the MoPOT2 repetitive element. CK, rice leaf segments inoculated with agar plugs without fungal mycelium. (C) Rice root infection assay. Arrows indicate typical necrotic lesions. (D) qPCR analysis of fungal biomass in inoculated rice roots. (E) Appressorium development on hydrophobic coverslip. Scale bar: 10 μm.
    Figure Legend Snippet: MoSNT2 is essential for plant infection by M. oryzae . (A) Rice leaf segments infected with fungal mycelium. (B) Measurement of fungal biomass in infected leaves based on qPCR analysis of the MoPOT2 repetitive element. CK, rice leaf segments inoculated with agar plugs without fungal mycelium. (C) Rice root infection assay. Arrows indicate typical necrotic lesions. (D) qPCR analysis of fungal biomass in inoculated rice roots. (E) Appressorium development on hydrophobic coverslip. Scale bar: 10 μm.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    MoSNT2 regulates cell wall integrity and oxidative stress response. (A) Growth of M. oryzae on CM agar medium containing 200 μg/ml CFW or 200 μg/ml CR for 5 days. (B) CFW staining and epifluorescence microscopy of cell wall chitin of mycelium grown in liquid CM. Scale bar: 20 μm. (C) Increased hyphal melanization as a consequence of MoSNT2 deletion. (D) qRT-PCR analysis on the expression levels of melanin biosynthesis genes in mycelium grown in liquid CM. (E) Mycelial growth on CM agar medium in the presence of different concentrations of H 2 O 2 . (F) Statistical analysis of the inhibition rate under H 2 O 2 -induced oxidative stress on mycelial growth. Asterisks represent significant differences (** PÂÂ
    Figure Legend Snippet: MoSNT2 regulates cell wall integrity and oxidative stress response. (A) Growth of M. oryzae on CM agar medium containing 200 μg/ml CFW or 200 μg/ml CR for 5 days. (B) CFW staining and epifluorescence microscopy of cell wall chitin of mycelium grown in liquid CM. Scale bar: 20 μm. (C) Increased hyphal melanization as a consequence of MoSNT2 deletion. (D) qRT-PCR analysis on the expression levels of melanin biosynthesis genes in mycelium grown in liquid CM. (E) Mycelial growth on CM agar medium in the presence of different concentrations of H 2 O 2 . (F) Statistical analysis of the inhibition rate under H 2 O 2 -induced oxidative stress on mycelial growth. Asterisks represent significant differences (** PÂÂ

    Techniques Used: Staining, Epifluorescence Microscopy, Quantitative RT-PCR, Expressing, Inhibition

    17) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.
    Figure Legend Snippet: MoSNT2 plays critical roles in autophagy of M. oryzae . (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.

    Techniques Used: Expressing, Staining, Fluorescence

    18) Product Images from "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning"

    Article Title: A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning

    Journal: Nature protocols

    doi: 10.1038/nprot.2008.250

    Strategy to clone the engineered synthetic silk-like sequences in the pET-19b expression vector. The black stars (★) indicate the restriction digestion of DNA.
    Figure Legend Snippet: Strategy to clone the engineered synthetic silk-like sequences in the pET-19b expression vector. The black stars (★) indicate the restriction digestion of DNA.

    Techniques Used: Positron Emission Tomography, Expressing, Plasmid Preparation

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