Journal: Journal of Clinical Investigation

Article Title: SF3B1 mutation and ATM deletion codrive leukemogenesis via centromeric R-loop dysregulation

doi: 10.1172/jci163325

Figure Lengend Snippet: Figure 3. SF3B1 mutation–associated cen-R-loop dysregulation leads to aneuploidy. (A–C) Analysis of 2-dimensional cross-sectional area of the entire body of chromosomes (A) and aberrant mitosis frequency (B) and spindle length and width (C) in HEK293T SF3B1 WT and MT overexpressing either empty vector (EV) or WT RNH1. (D) DRIP-qPCR in HEK293T SF3B1 WT and MT overexpressing either dCas9–GFP–RNaseH1 WKKD (WKKD RNH1) or dCas9–GFP–RNaseH1 WT (WT RNH1) in combination with either sgRNA guide control (sgCTRL) or sgRNA targeting α-satellite centromeric repeats (sgAlphaSat). Centromeric arrays: The chromosome is specified by the number following the “D”; and the array is specified by the number following the “Z.” Two-way ANOVA test. (E and F) Analysis of 2-dimensional cross-sectional area of the entire body of chromosomes (E) and aberrant mitosis fre- quency (F) in cells from D. Box plots show the median and 25th and 75th percentiles, with whiskers extending to minimum and maximum values. Bar graphs represent mean ± SD. Each dot represents a biological replicate. Two-tailed unpaired t test followed by Bonferroni’s post hoc test, except in D.

Article Snippet: RNaseH1 WKKD–eGFP–dCas9 construct was generated by introduction of point mutations to dRNaseH1-eGFP-dCas9 (139836, Addgene) construct using the Q5 site-directed mutagenesis kit (New England Biolabs) according to the manufacturer’s protocol with minor modification (15 minutes of incubation with KLD enzyme mix).

Techniques: Mutagenesis, Plasmid Preparation, Control, Two Tailed Test

Journal: Frontiers in Plant Science

Article Title: Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

doi: 10.3389/fpls.2017.01567

Figure Lengend Snippet: Yeast expression plasmids and yeast strains used in this work.

Article Snippet: E. coli was grown at 37°C in LB medium supplemented with appropriate antibiotics. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were PCR amplified using primers 5′-AAAAGGATCCAATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTCACGTGTTAATGGTGATGATGGTGGTG-3′, with pN2SB22 and pN2SB24 as templates, respectively. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were digested with BamH I and Pml I, and inserted into pGFPGUSPlus vector ( ) (Addgene plasmid #64401) digested with Bgl II and Pml I, replacing GUS and generating pN2XJ13 ( su9-nifB Av -His 10 ) and pN2XJ14 ( su9-nifB Mi -His 10 ), respectively. su9-nifB Av was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCCTTAGCTTGCAACAAAGC-3′, with pN2SB22 as template. su9-nifB Av was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ15 for expression of su9-nifB Av -gfp. su9-nifB Mi was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCGTGTGAGAAATGCTTCAAGTCG-3′, with pN2SB24 as template. su9-nifB Mi was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ16 for expression of su9-nifB Mi -gfp .

Techniques: Expressing, Transformation Assay

Journal: Frontiers in Plant Science

Article Title: Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

doi: 10.3389/fpls.2017.01567

Figure Lengend Snippet: Purification and biochemical properties of yNifB Mi . (A) SDS-PAGE and Western blot analysis of yNifB Mi purification. CFE, 65°C heated SB10Y cell-free extract; FT, affinity chromatography flow through; W1-W4 and E1-E2, affinity chromatography wash and elution fractions containing increasing concentrations of imidazole (see Materials and Methods for details). Grey arrow in the Coomassie stained panel points to the position of yNifB Mi in the gel. (B) SU9 processing site (black arrow) of yNifB Mi . Underlined sequence indicates the N-terminal amino acids of yNifB Mi identified by Edman degradation. (C) UV-visible spectra of as isolated, reconstituted, and dithionite (DTH)-reduced reconstituted yNifB Mi . (D) Typical color of as isolated and reconstituted yNifB Mi purified preparations. (E) Titration of FeMo-co synthesis and nitrogenase reconstitution assay with yNifB Mi . The indicated concentrations of yNifB Mi monomer were used. NifB activity was determined by acetylene reduction assay of reconstituted NifDK from ΔnifB A. vinelandii UW140 cell-free extracts. Data represent mean ± standard deviation ( n = 2) at each yNifB Mi concentration.

Article Snippet: E. coli was grown at 37°C in LB medium supplemented with appropriate antibiotics. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were PCR amplified using primers 5′-AAAAGGATCCAATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTCACGTGTTAATGGTGATGATGGTGGTG-3′, with pN2SB22 and pN2SB24 as templates, respectively. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were digested with BamH I and Pml I, and inserted into pGFPGUSPlus vector ( ) (Addgene plasmid #64401) digested with Bgl II and Pml I, replacing GUS and generating pN2XJ13 ( su9-nifB Av -His 10 ) and pN2XJ14 ( su9-nifB Mi -His 10 ), respectively. su9-nifB Av was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCCTTAGCTTGCAACAAAGC-3′, with pN2SB22 as template. su9-nifB Av was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ15 for expression of su9-nifB Av -gfp. su9-nifB Mi was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCGTGTGAGAAATGCTTCAAGTCG-3′, with pN2SB24 as template. su9-nifB Mi was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ16 for expression of su9-nifB Mi -gfp .

Techniques: Purification, SDS Page, Western Blot, Affinity Chromatography, Staining, Sequencing, Isolation, Titration, Reconstitution Assay, Activity Assay, Acetylene Reduction Assay, Standard Deviation, Concentration Assay

Journal: Frontiers in Plant Science

Article Title: Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

doi: 10.3389/fpls.2017.01567

Figure Lengend Snippet: Expression of mitochondria targeted (SU9) NifB Av and NifB Mi GFP fusions in N. benthamiana leaves. (A,B) Mesophyll cells expressing SU9-NifB Av -GFP (A) or SU9-NifB Mi -GFP (B). GFP (green) and chlorophyll autofluorescence (red) of chloroplasts is shown. (C–E) Epidermal cells expressing SU9-NifB Av -GFP (C) and SU9-NifB Mi -GFP (D,E), together with a fluorescent mitochondria marker (Mito-RFP). GFP (green), Mito-RFP (magenta) and chlorophyll autofluorescence (red) of chloroplasts is shown. Co-localization of SU9-NifB Av -GFP or SU9-NifB Mi -GFP constructs with Mito-RFP labeled structures is shown as white in the merged images, and highlighted with yellow arrows. Adjacent cells expressing SU9-NifB Mi -GFP or Mito-RFP are shown as control to verify the specificity of the signal recorded in each channel (E). Scale bars show 30 μm. Confocal Microscopy conditions are specified in Materials and Methods.

Article Snippet: E. coli was grown at 37°C in LB medium supplemented with appropriate antibiotics. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were PCR amplified using primers 5′-AAAAGGATCCAATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTCACGTGTTAATGGTGATGATGGTGGTG-3′, with pN2SB22 and pN2SB24 as templates, respectively. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were digested with BamH I and Pml I, and inserted into pGFPGUSPlus vector ( ) (Addgene plasmid #64401) digested with Bgl II and Pml I, replacing GUS and generating pN2XJ13 ( su9-nifB Av -His 10 ) and pN2XJ14 ( su9-nifB Mi -His 10 ), respectively. su9-nifB Av was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCCTTAGCTTGCAACAAAGC-3′, with pN2SB22 as template. su9-nifB Av was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ15 for expression of su9-nifB Av -gfp. su9-nifB Mi was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCGTGTGAGAAATGCTTCAAGTCG-3′, with pN2SB24 as template. su9-nifB Mi was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ16 for expression of su9-nifB Mi -gfp .

Techniques: Expressing, Marker, Construct, Labeling, Confocal Microscopy

Journal: Frontiers in Plant Science

Article Title: Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

doi: 10.3389/fpls.2017.01567

Figure Lengend Snippet: Expression and solubility of mitochondria targeted (SU9) NifB Av and NifB Mi in N. benthamiana leaves. (A) Western blot analysis of total protein extracts (TE) prepared from infiltrated N. benthamiana leaves expressing GFP, SU9-NifB Av -GFP or SU9-NifB Mi -GFP. Blue arrows indicate the polypeptide recognized both by GFP and NifB Av specific antibodies. Short (s.e.) and long (l.e.) film exposures of the GFP antibody probed membrane are shown. (B) Migration of SU9-NifB Av -His 10 when expressed in S. cerevisiae and N. benthamiana . Migration in SDS-PAGE was determined after Western blot analysis using NifB Av specific antibodies. Total protein extracts (TE) from W303-1a S. cerevisiae cells (WT) or cells expressing SU9-NifB Av -His 10 (SB09Y) were prepared. Soluble protein extracts (S) from N. benthamiana leaf cells infiltrated with A. tumefaciens containing control vector (pGFPGUSPlus) or vector for expression of SU9-NifB Av -His 10 (pN2XJ13). Dotted line indicate different exposures of the right part of the membrane. See Supplementary Figure for entire gel of the cropped exposure. (C) Migration of SU9-NifB Mi -His 10 when expressed in S. cerevisiae and N. benthamiana . Migration in SDS-PAGE was determined after Western blot analysis using NifB Mi specific antibodies. Total protein extracts (TE) from W303-1a S. cerevisiae cells (WT) or cells expressing SU9-NifB Mi -His 10 (SB10Y) were prepared. Soluble protein extracts (S) from N. benthamiana leaf cells infiltrated with A. tumefaciens containing control vector (pGFPGUSPlus) or vector for expression of SU9-NifB Mi -His 10 (pN2XJ14). As control of N. benthamiana leaf infiltration, GFP expressed from the pGFPGUSPlus vector backbone was detected (B,C).

Article Snippet: E. coli was grown at 37°C in LB medium supplemented with appropriate antibiotics. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were PCR amplified using primers 5′-AAAAGGATCCAATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTCACGTGTTAATGGTGATGATGGTGGTG-3′, with pN2SB22 and pN2SB24 as templates, respectively. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were digested with BamH I and Pml I, and inserted into pGFPGUSPlus vector ( ) (Addgene plasmid #64401) digested with Bgl II and Pml I, replacing GUS and generating pN2XJ13 ( su9-nifB Av -His 10 ) and pN2XJ14 ( su9-nifB Mi -His 10 ), respectively. su9-nifB Av was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCCTTAGCTTGCAACAAAGC-3′, with pN2SB22 as template. su9-nifB Av was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ15 for expression of su9-nifB Av -gfp. su9-nifB Mi was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCGTGTGAGAAATGCTTCAAGTCG-3′, with pN2SB24 as template. su9-nifB Mi was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ16 for expression of su9-nifB Mi -gfp .

Techniques: Expressing, Solubility, Western Blot, Migration, SDS Page, Plasmid Preparation

Journal: Frontiers in Plant Science

Article Title: Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

doi: 10.3389/fpls.2017.01567

Figure Lengend Snippet: Tobacco expressed nitrogenase related proteins and their expected sizes.

Article Snippet: E. coli was grown at 37°C in LB medium supplemented with appropriate antibiotics. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were PCR amplified using primers 5′-AAAAGGATCCAATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTCACGTGTTAATGGTGATGATGGTGGTG-3′, with pN2SB22 and pN2SB24 as templates, respectively. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were digested with BamH I and Pml I, and inserted into pGFPGUSPlus vector ( ) (Addgene plasmid #64401) digested with Bgl II and Pml I, replacing GUS and generating pN2XJ13 ( su9-nifB Av -His 10 ) and pN2XJ14 ( su9-nifB Mi -His 10 ), respectively. su9-nifB Av was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCCTTAGCTTGCAACAAAGC-3′, with pN2SB22 as template. su9-nifB Av was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ15 for expression of su9-nifB Av -gfp. su9-nifB Mi was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCGTGTGAGAAATGCTTCAAGTCG-3′, with pN2SB24 as template. su9-nifB Mi was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ16 for expression of su9-nifB Mi -gfp .

Techniques:

Journal: Frontiers in Plant Science

Article Title: Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

doi: 10.3389/fpls.2017.01567

Figure Lengend Snippet: Expression and solubility of mitochondria targeted (COX4) NifB Av and NifB Mi in N. benthamiana leaves. (A) Western blot analysis of total protein extracts (TE) prepared from infiltrated N. benthamiana leaves expressing COX4-twinStrep-GFP (GFP), COX4-twinStrep-NifB Av (NifB Av ) or COX4-twinStrep-NifB Mi (NifB Mi ) and separated by SDS-PAGE. The COX4-twinStrep-GFP (green arrow), COX4-twinStrep-NifB Av (blue arrow), COX4-twinStrep-NifB Mi (red arrow) proteins are highlighted. A pronounced non-specific polypeptide detected using the Strep-tag antibodies (white star) co-migrated with the large subunit of Rubisco. The membrane probed with antibodies against Rubisco was also stained with Ponceau and is included as loading control. (B,C) Western blot analysis of the soluble (S) and non-soluble pellet (P) fractions of N. benthamiana leaf total extracts used in (A), using Strep-tag antibodies (B) or NifB Av antibodies (C). The COX4-twinStrep-GFP (green arrow), COX4-twinStrep-NifB Av (blue arrow), COX4-twinStrep-NifB Mi (red arrow) proteins are highlighted. Non-specific bands detected using the Strep-tag antibodies (white stars) co-migrated with Rubisco (B). Non-specific bands detected with NifB Av antibodies (black stars) are also indicated (C). Short (s.e.) and long (l.e.) film exposures of the Strep-tag antibody probed membrane are shown (B). Ponceau staining of the NifB Av antibody probed membrane is shown as loading control (C).

Article Snippet: E. coli was grown at 37°C in LB medium supplemented with appropriate antibiotics. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were PCR amplified using primers 5′-AAAAGGATCCAATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTCACGTGTTAATGGTGATGATGGTGGTG-3′, with pN2SB22 and pN2SB24 as templates, respectively. su9-nifB Av -His 10 and su9-nifB Mi -His 10 were digested with BamH I and Pml I, and inserted into pGFPGUSPlus vector ( ) (Addgene plasmid #64401) digested with Bgl II and Pml I, replacing GUS and generating pN2XJ13 ( su9-nifB Av -His 10 ) and pN2XJ14 ( su9-nifB Mi -His 10 ), respectively. su9-nifB Av was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCCTTAGCTTGCAACAAAGC-3′, with pN2SB22 as template. su9-nifB Av was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ15 for expression of su9-nifB Av -gfp. su9-nifB Mi was PCR amplified using primers 5′-AAAAGCTAGCATGGCCTCCACTCGTGTCCTCG-3′ and 5′-TTTTGCTAGCGCGTGTGAGAAATGCTTCAAGTCG-3′, with pN2SB24 as template. su9-nifB Mi was digested with Nhe I and inserted into pGFPGUSPlus vector digested with Xba I, generating pN2XJ16 for expression of su9-nifB Mi -gfp .

Techniques: Expressing, Solubility, Western Blot, SDS Page, Strep-tag, Staining