polypyrimidine tract binding protein ptb  (New England Biolabs)


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    Gibson Assembly Master Mix
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    Gibson Assembly Master Mix 50 rxns
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    New England Biolabs polypyrimidine tract binding protein ptb
    Gibson Assembly Master Mix
    Gibson Assembly Master Mix 50 rxns
    https://www.bioz.com/result/polypyrimidine tract binding protein ptb/product/New England Biolabs
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    Images

    1) Product Images from "GoldCLIP: Gel-omitted Ligation-dependent CLIP"

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    Journal: Genomics, Proteomics & Bioinformatics

    doi: 10.1016/j.gpb.2018.04.003

    HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.
    Figure Legend Snippet: HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Techniques Used: Flow Cytometry, Stable Transfection, Expressing, Irradiation, Lysis, Magnetic Beads, Purification, Clone Assay, Next-Generation Sequencing, Western Blot, Transfection, Staining, RNA Binding Assay, Cross-linking Immunoprecipitation, Binding Assay

    2) Product Images from "CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3"

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3

    Journal: EvoDevo

    doi: 10.1186/s13227-017-0073-y

    CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif
    Figure Legend Snippet: CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

    Techniques Used: CRISPR, Plasmid Preparation, Modification, Polymerase Chain Reaction, Sequencing

    Tribolium Robo2/3 can substitute for Drosophila Robo3 to promote axon pathway formation in Drosophila embryos. a – d Stage 16 Drosophila embryos stained with anti-HRP ( magenta ) and anti-FasII ( green ) antibodies. Lower images show anti-FasII channel alone from the same embryos. In wild-type embryos, FasII-positive axons form three distinct longitudinal pathways on either side of the midline, one each in the medial, intermediate, and lateral zones of the neuropile. The intermediate FasII pathway is distinct from the medial and lateral pathways in every hemisegment in wild-type embryos ( a , arrow ). In robo3 1 embryos, FasII-positive axons that normally form the intermediate pathway are displaced medially, and the intermediate pathway fails to form ( b , arrow with asterisk ). Intermediate pathways form correctly in embryos in which the robo3 gene is replaced with a robo3 cDNA ( c , arrow ). When robo3 is replaced with a TcRobo2/3 cDNA, intermediate pathways form correctly in over 88% of hemisegments ( d , arrow ), indicating that TcRobo2/3 can substitute for robo3 to promote axon pathway formation in the intermediate region of the neuropile. Bar graph shows quantification of intermediate FasII pathway defects in the genotypes shown in a – d . Error bars indicate standard error of the mean. Number of embryos scored for each genotype is indicated in parentheses . e – h Embryos carrying the sema2b - TauMyc transgene and stained with anti-HRP ( blue ), anti-FasII ( red ), and anti-Myc ( green ) antibodies. The sema2b - TauMyc transgene labels the cell bodies and axons of 2–3 neurons per hemisegment in abdominal segments A4–A8. These axons normally project across the midline and then extend anteriorly in the intermediate region of the neuropile ( e , arrowhead ). In robo3 1 embryos, these axons are displaced medially ( f , arrowhead with asterisk ), but their normal intermediate position is restored in both robo3 robo3 ( g , arrowhead ) and robo3 TcRobo2/3 embryos ( h , arrowhead )
    Figure Legend Snippet: Tribolium Robo2/3 can substitute for Drosophila Robo3 to promote axon pathway formation in Drosophila embryos. a – d Stage 16 Drosophila embryos stained with anti-HRP ( magenta ) and anti-FasII ( green ) antibodies. Lower images show anti-FasII channel alone from the same embryos. In wild-type embryos, FasII-positive axons form three distinct longitudinal pathways on either side of the midline, one each in the medial, intermediate, and lateral zones of the neuropile. The intermediate FasII pathway is distinct from the medial and lateral pathways in every hemisegment in wild-type embryos ( a , arrow ). In robo3 1 embryos, FasII-positive axons that normally form the intermediate pathway are displaced medially, and the intermediate pathway fails to form ( b , arrow with asterisk ). Intermediate pathways form correctly in embryos in which the robo3 gene is replaced with a robo3 cDNA ( c , arrow ). When robo3 is replaced with a TcRobo2/3 cDNA, intermediate pathways form correctly in over 88% of hemisegments ( d , arrow ), indicating that TcRobo2/3 can substitute for robo3 to promote axon pathway formation in the intermediate region of the neuropile. Bar graph shows quantification of intermediate FasII pathway defects in the genotypes shown in a – d . Error bars indicate standard error of the mean. Number of embryos scored for each genotype is indicated in parentheses . e – h Embryos carrying the sema2b - TauMyc transgene and stained with anti-HRP ( blue ), anti-FasII ( red ), and anti-Myc ( green ) antibodies. The sema2b - TauMyc transgene labels the cell bodies and axons of 2–3 neurons per hemisegment in abdominal segments A4–A8. These axons normally project across the midline and then extend anteriorly in the intermediate region of the neuropile ( e , arrowhead ). In robo3 1 embryos, these axons are displaced medially ( f , arrowhead with asterisk ), but their normal intermediate position is restored in both robo3 robo3 ( g , arrowhead ) and robo3 TcRobo2/3 embryos ( h , arrowhead )

    Techniques Used: Staining

    TcRobo2/3 expression reproduces Robo3’s expression pattern in the robo3 TcRobo2/3 allele. a – d Stage 16 Drosophila embryos stained with anti-HRP ( magenta ; labels all axons) and anti-Robo3 ( green ) antibodies. Lower images show anti-Robo3 channel alone from the same embryos. In wild-type embryos, endogenous Robo3 protein is detectable on longitudinal axons within the outer two-thirds of the neuropile ( a , arrowhead ). Robo3 protein is undetectable in embryos homozygous for the loss of function robo3 1 allele ( b , arrowhead with asterisk ) [ 7 , 8 ]. There are no large-scale defects detectable with anti-HRP in the axon scaffold of robo3 1 mutants. In embryos in which the robo3 gene has been replaced with an HA-tagged robo3 cDNA, Robo3 protein expressed from the modified locus reproduces its normal expression pattern ( c , arrowhead ) [ 8 ]. In our CRISPR-modified embryos in which robo3 has been replaced by TcRobo2/3 , Robo3 protein is undetectable, consistent with the removal of robo3 coding sequences ( d , arrowhead with asterisk ). e , f Stage 16 embryos stained with anti-HRP ( magenta ) and anti-HA ( green ) antibodies. Lower images show anti-HA channel alone from the same embryos. Anti-HA staining in robo3 robo3 embryos detects the Robo3 protein expressed from the modified locus and reproduces the staining pattern seen with anti-Robo3 ( e , arrowhead ). In robo3 TcRobo2/3 embryos, the HA-tagged TcRobo2/3 protein reproduces Robo3’s expression pattern and is detectable on longitudinal axons within the lateral two-thirds of the neuropile ( f , arrowhead ). Schematics of the two modified robo3 alleles are shown at lower left . The robo3 robo3 allele was generated by Spitzweck et al. [ 8 ]
    Figure Legend Snippet: TcRobo2/3 expression reproduces Robo3’s expression pattern in the robo3 TcRobo2/3 allele. a – d Stage 16 Drosophila embryos stained with anti-HRP ( magenta ; labels all axons) and anti-Robo3 ( green ) antibodies. Lower images show anti-Robo3 channel alone from the same embryos. In wild-type embryos, endogenous Robo3 protein is detectable on longitudinal axons within the outer two-thirds of the neuropile ( a , arrowhead ). Robo3 protein is undetectable in embryos homozygous for the loss of function robo3 1 allele ( b , arrowhead with asterisk ) [ 7 , 8 ]. There are no large-scale defects detectable with anti-HRP in the axon scaffold of robo3 1 mutants. In embryos in which the robo3 gene has been replaced with an HA-tagged robo3 cDNA, Robo3 protein expressed from the modified locus reproduces its normal expression pattern ( c , arrowhead ) [ 8 ]. In our CRISPR-modified embryos in which robo3 has been replaced by TcRobo2/3 , Robo3 protein is undetectable, consistent with the removal of robo3 coding sequences ( d , arrowhead with asterisk ). e , f Stage 16 embryos stained with anti-HRP ( magenta ) and anti-HA ( green ) antibodies. Lower images show anti-HA channel alone from the same embryos. Anti-HA staining in robo3 robo3 embryos detects the Robo3 protein expressed from the modified locus and reproduces the staining pattern seen with anti-Robo3 ( e , arrowhead ). In robo3 TcRobo2/3 embryos, the HA-tagged TcRobo2/3 protein reproduces Robo3’s expression pattern and is detectable on longitudinal axons within the lateral two-thirds of the neuropile ( f , arrowhead ). Schematics of the two modified robo3 alleles are shown at lower left . The robo3 robo3 allele was generated by Spitzweck et al. [ 8 ]

    Techniques Used: Expressing, Staining, Modification, CRISPR, Generated

    Sequence comparison of Drosophila Robo3 and Tribolium Robo2/3. a Schematic comparison of the two receptors showing conserved domain structure and percent identity between individual ectodomain elements. The highest degree of sequence conservation occurs within the Slit-binding Ig1 domain (70% identity). While both proteins share the evolutionarily conserved CC0 and CC1 motifs, the TcRobo2/3 cytodomain (206 aa) is less than half the length of the Robo3 cytodomain (452 aa). b Protein sequence alignment. Structural features are indicated below the sequence. Fn domains have been re-annotated relative to Evans and Bashaw [ 11 ] based on revised predictions of beta strand locations. Identical residues are shaded black ; similar residues are shaded gray . Ig immunoglobulin-like domain, Fn fibronectin type III repeat, Tm transmembrane helix, CC conserved cytoplasmic motif
    Figure Legend Snippet: Sequence comparison of Drosophila Robo3 and Tribolium Robo2/3. a Schematic comparison of the two receptors showing conserved domain structure and percent identity between individual ectodomain elements. The highest degree of sequence conservation occurs within the Slit-binding Ig1 domain (70% identity). While both proteins share the evolutionarily conserved CC0 and CC1 motifs, the TcRobo2/3 cytodomain (206 aa) is less than half the length of the Robo3 cytodomain (452 aa). b Protein sequence alignment. Structural features are indicated below the sequence. Fn domains have been re-annotated relative to Evans and Bashaw [ 11 ] based on revised predictions of beta strand locations. Identical residues are shaded black ; similar residues are shaded gray . Ig immunoglobulin-like domain, Fn fibronectin type III repeat, Tm transmembrane helix, CC conserved cytoplasmic motif

    Techniques Used: Sequencing, Binding Assay

    3) Product Images from "Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways"

    Article Title: Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25356

    Melanoma-common PAK5 mutants do not alter kinase activity A. Protein diagram depicting the location of human PAK5 variants examined in this study. Dots are color-coordinated to divide the mutations into groups: the most frequent melanoma variants (E144K, M173I, E294K, S364L and D421N) in blue, a kinase dead mutant (K478/9R, KD) in red, and a constitutive active mutant (S573N, CA) in green. PBD = p21-binding domain, PS/AID = pseudosubstrate/auto-inhibitory domain, Kinase = kinase domain. Orange boxes represent proline-rich motifs (PxxP). B. Immunoblot showing the stable expression of FLAG-tagged PAK5 variants in immortalized human primary melanocytes (hMELTs). ‘hMELT’ indicates the parental cell line. Shown below each lane is the β-ACTIN-normalized level of mutant PAK5 expression relative to PAK5 wildtype (WT). C. PAK5 kinase activity was measured using the ADP-Glo system and normalized to immunoprecipitated protein levels as measured by immunoblot. A representative immunoblot of immunoprecipitated FLAG-PAK5 is shown. Each bar represents three biological replicates with the mean and standard deviation indicated. † = p
    Figure Legend Snippet: Melanoma-common PAK5 mutants do not alter kinase activity A. Protein diagram depicting the location of human PAK5 variants examined in this study. Dots are color-coordinated to divide the mutations into groups: the most frequent melanoma variants (E144K, M173I, E294K, S364L and D421N) in blue, a kinase dead mutant (K478/9R, KD) in red, and a constitutive active mutant (S573N, CA) in green. PBD = p21-binding domain, PS/AID = pseudosubstrate/auto-inhibitory domain, Kinase = kinase domain. Orange boxes represent proline-rich motifs (PxxP). B. Immunoblot showing the stable expression of FLAG-tagged PAK5 variants in immortalized human primary melanocytes (hMELTs). ‘hMELT’ indicates the parental cell line. Shown below each lane is the β-ACTIN-normalized level of mutant PAK5 expression relative to PAK5 wildtype (WT). C. PAK5 kinase activity was measured using the ADP-Glo system and normalized to immunoprecipitated protein levels as measured by immunoblot. A representative immunoblot of immunoprecipitated FLAG-PAK5 is shown. Each bar represents three biological replicates with the mean and standard deviation indicated. † = p

    Techniques Used: Activity Assay, Mutagenesis, Binding Assay, Expressing, Immunoprecipitation, Standard Deviation

    Melanoma-associated PAK5 mutants do not affect in vitro melanocyte migration or resistance to temozolomide A. hMELT stable cell lines were placed in the seeding port of the depicted microfluidics device. These cells were serum-starved while full serum medium was added to the flanking collection ports to stimulate migration. Using time-lapse microscopy, images were taken every five minutes for a total of twelve hours. Shown is a representative image of cell tracking through the microfluidics device channels using ImageJ. B. The average speed (µm/min) at which hMELT stable cell lines traversed the microfluidics device is shown relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). C. The ability of single cells to persistently migrate in one direction is shown for each PAK5 mutant relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). D. hMELT parental and wildtype (WT) PAK5 cells were seeded in a 96-well plate and treated with various concentrations of temozolomide ranging from 0 µM to 1 mM. Treatment media was changed every two days for a total of seven days after which cell viability was measured using resazurin metabolism. The IC 50 was determined using the ‘log(inhibitor) vs. response (three parameters)’ analysis in GraphPad Prism software. E. hMELT-PAK5 stable cell lines were seeded in a 96-well plate and treated with either 250 µM temozolomide or DMSO. The treatment media was changed every two days for a total of seven days. Cell viability was measured using resazurin metabolism and normalized to the DMSO control. ‘hMELT’ indicates the parental cell line. Each bar represents five biological replicates performed in triplicate with the mean and standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%).
    Figure Legend Snippet: Melanoma-associated PAK5 mutants do not affect in vitro melanocyte migration or resistance to temozolomide A. hMELT stable cell lines were placed in the seeding port of the depicted microfluidics device. These cells were serum-starved while full serum medium was added to the flanking collection ports to stimulate migration. Using time-lapse microscopy, images were taken every five minutes for a total of twelve hours. Shown is a representative image of cell tracking through the microfluidics device channels using ImageJ. B. The average speed (µm/min) at which hMELT stable cell lines traversed the microfluidics device is shown relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). C. The ability of single cells to persistently migrate in one direction is shown for each PAK5 mutant relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). D. hMELT parental and wildtype (WT) PAK5 cells were seeded in a 96-well plate and treated with various concentrations of temozolomide ranging from 0 µM to 1 mM. Treatment media was changed every two days for a total of seven days after which cell viability was measured using resazurin metabolism. The IC 50 was determined using the ‘log(inhibitor) vs. response (three parameters)’ analysis in GraphPad Prism software. E. hMELT-PAK5 stable cell lines were seeded in a 96-well plate and treated with either 250 µM temozolomide or DMSO. The treatment media was changed every two days for a total of seven days. Cell viability was measured using resazurin metabolism and normalized to the DMSO control. ‘hMELT’ indicates the parental cell line. Each bar represents five biological replicates performed in triplicate with the mean and standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%).

    Techniques Used: In Vitro, Migration, Stable Transfection, Time-lapse Microscopy, Cell Tracking Assay, Mutagenesis, Software

    PAK5 S364L and D421N promote melanocyte proliferation and ERK activation A. hMELT stable cell lines were placed in 2% serum for 18 hours and then labeled with EdU for eight hours. After labeling, cells were fixed for flow cytometric analysis. Shown is the average percentage of EdU positive cells across at least three biological replicates with error bars representing the standard deviation. † = p
    Figure Legend Snippet: PAK5 S364L and D421N promote melanocyte proliferation and ERK activation A. hMELT stable cell lines were placed in 2% serum for 18 hours and then labeled with EdU for eight hours. After labeling, cells were fixed for flow cytometric analysis. Shown is the average percentage of EdU positive cells across at least three biological replicates with error bars representing the standard deviation. † = p

    Techniques Used: Activation Assay, Stable Transfection, Labeling, Flow Cytometry, Standard Deviation

    PAK5 S364L and D421N promote proliferation through PKA activation A. Lysates from hMELT stable cell lines, incubated in 2% serum for 18 hours, were analyzed by SDS-PAGE followed by immunoblotting with an antibody that recognizes the phosphorylated substrate motif of AGC kinases. ‘hMELT’ indicates the parental cell line. Each bar indicates the average, β-ACTIN-normalized, pAGC substrate signal relative to parental hMELT cells across at least three biological replicates. Error bars represent the standard deviation. † = p
    Figure Legend Snippet: PAK5 S364L and D421N promote proliferation through PKA activation A. Lysates from hMELT stable cell lines, incubated in 2% serum for 18 hours, were analyzed by SDS-PAGE followed by immunoblotting with an antibody that recognizes the phosphorylated substrate motif of AGC kinases. ‘hMELT’ indicates the parental cell line. Each bar indicates the average, β-ACTIN-normalized, pAGC substrate signal relative to parental hMELT cells across at least three biological replicates. Error bars represent the standard deviation. † = p

    Techniques Used: Activation Assay, Stable Transfection, Incubation, SDS Page, Standard Deviation

    PAK5 is the most frequently altered PAK family member in melanoma A. Oncoprint generated in cBioPortal depicting alterations in the PAK gene family for 287 melanomas collected by TCGA (provisional data accessed on 08/2016). PAKs are divided into two groups based upon protein sequence homology: group I (PAK1, PAK2 and PAK3) and group II (PAK4, PAK5, and PAK6). B. Protein diagram of human PAK5 depicting the location and frequency of melanoma-associated variants. Green dots represent missense mutations, black dots represent truncating mutations, and purple dots represent splice-site mutations.
    Figure Legend Snippet: PAK5 is the most frequently altered PAK family member in melanoma A. Oncoprint generated in cBioPortal depicting alterations in the PAK gene family for 287 melanomas collected by TCGA (provisional data accessed on 08/2016). PAKs are divided into two groups based upon protein sequence homology: group I (PAK1, PAK2 and PAK3) and group II (PAK4, PAK5, and PAK6). B. Protein diagram of human PAK5 depicting the location and frequency of melanoma-associated variants. Green dots represent missense mutations, black dots represent truncating mutations, and purple dots represent splice-site mutations.

    Techniques Used: Generated, Sequencing

    4) Product Images from "Mixed Culture of Bacterial Cell for Large Scale DNA Storage"

    Article Title: Mixed Culture of Bacterial Cell for Large Scale DNA Storage

    Journal: bioRxiv

    doi: 10.1101/2020.02.21.960476

    A large-scale DNA data storage in living cell. a) The workflow for the manufacture of mixed culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to assembly and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original master pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.
    Figure Legend Snippet: A large-scale DNA data storage in living cell. a) The workflow for the manufacture of mixed culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to assembly and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original master pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.

    Techniques Used: Transformation Assay, Amplification, Plasmid Preparation, Passaging, Sequencing

    5) Product Images from "Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference"

    Article Title: Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw063

    The roX1 expression is efficiently silenced by CRISPRi. ( A ) Schematic representation of the Drosophila roX1 locus and the relative positions of the roX1 -targeting sgRNAs. There are two transcription start sites of roX1 gene (270 nt apart) which produce isoforms RA and RB (FlyBase). The sgRNAs targeting the RNA polymerase template (rT, red) and non-template (rNT, violet) strands are shown. The 5′ intergenic region between roX1 RA and the upstream gene yin is shown in solid black line. The arrowheads mark the position of the primers used to detect roX1 RNA (black) and roX1 RA isoform (blue) for RT-qPCR analysis. ( B ) RT-PCR analysis using clone 8 cells shows the presence of roX1 RA and RB transcripts. The primers specific to sequences in the intergenic region, roX1 RA and roX1 RB isoform were used for PCR amplification. ( C, D, E ) RT-qPCR measurement of abundance of the roX1 and roX2 transcripts in cell lines coexpressing human (C, D) or Drosophila (E) dCas9 protein and sgRNAs (as shown on the x-axis) complementary to the rT or rNT DNA strand at the endogenous roX1 locus. The cell lines expressing single sgRNA (C), or two guide RNAs simultaneously (D, E) are shown. The gRNAs rNT7, rT8 and rT9 mediate efficient knockdown of the roX1 transcript, either alone or jointly with others. However, the combinations of rT1+rT3 and rT4+rNT5 which target the roX1 RA transcription initiation site, are effective only in pairs in the presence of human dCas9 and Drosophila dCas9, respectively. The y-axis shows the enrichment of RNAs relative to rp49 transcript and normalized to the cells transfected with pGTL-1 containing a non-targeting sequence (Ctrl). The data are shown from two biological replicates, each performed in triplicate. The error bars indicate SEM. The % shows knockdown in percentage as compared with the Ctrl.
    Figure Legend Snippet: The roX1 expression is efficiently silenced by CRISPRi. ( A ) Schematic representation of the Drosophila roX1 locus and the relative positions of the roX1 -targeting sgRNAs. There are two transcription start sites of roX1 gene (270 nt apart) which produce isoforms RA and RB (FlyBase). The sgRNAs targeting the RNA polymerase template (rT, red) and non-template (rNT, violet) strands are shown. The 5′ intergenic region between roX1 RA and the upstream gene yin is shown in solid black line. The arrowheads mark the position of the primers used to detect roX1 RNA (black) and roX1 RA isoform (blue) for RT-qPCR analysis. ( B ) RT-PCR analysis using clone 8 cells shows the presence of roX1 RA and RB transcripts. The primers specific to sequences in the intergenic region, roX1 RA and roX1 RB isoform were used for PCR amplification. ( C, D, E ) RT-qPCR measurement of abundance of the roX1 and roX2 transcripts in cell lines coexpressing human (C, D) or Drosophila (E) dCas9 protein and sgRNAs (as shown on the x-axis) complementary to the rT or rNT DNA strand at the endogenous roX1 locus. The cell lines expressing single sgRNA (C), or two guide RNAs simultaneously (D, E) are shown. The gRNAs rNT7, rT8 and rT9 mediate efficient knockdown of the roX1 transcript, either alone or jointly with others. However, the combinations of rT1+rT3 and rT4+rNT5 which target the roX1 RA transcription initiation site, are effective only in pairs in the presence of human dCas9 and Drosophila dCas9, respectively. The y-axis shows the enrichment of RNAs relative to rp49 transcript and normalized to the cells transfected with pGTL-1 containing a non-targeting sequence (Ctrl). The data are shown from two biological replicates, each performed in triplicate. The error bars indicate SEM. The % shows knockdown in percentage as compared with the Ctrl.

    Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Transfection, Sequencing

    CRISPRi in Drosophila . ( A ) Diagram showing the CRISPR interference (CRISPRi) system. To repress transcription, the catalytically inactive Cas9 protein (dCas9, green) is targeted either to the template or non-template DNA strand based on the targeting sequence of the sgRNA and an adjacent PAM sequence. Binding of the dCas9:sgRNA complex upstream of the transcription start site interferes with transcription initiation by preventing recruitment of the RNA polymerase while its assembly at a downstream site prevents transcription elongation. ( B ) Schematic representation of the transfection vectors pGTL-1 and pGTL-2. In pGTL-1, a single guide RNA with a 20 nt targeting sequence and the dCas9 protein are coexpressed under Drosophila constitutive promoters U6:3 and Actin5C , respectively. The dCas9 is separated from the blasticidin resistance gene (Bla R ) and eGFP by self-cleaving T2A peptides (dT2A and T2A). The guide RNA (gRNA) scaffold contains the U6 transcription terminator sequence. The pGTL-2 vector contains an additional sgRNA scaffold under the U6:1 promoter, thus allowing production of two sgRNAs simultaneously with the dCas9 protein. The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation.
    Figure Legend Snippet: CRISPRi in Drosophila . ( A ) Diagram showing the CRISPR interference (CRISPRi) system. To repress transcription, the catalytically inactive Cas9 protein (dCas9, green) is targeted either to the template or non-template DNA strand based on the targeting sequence of the sgRNA and an adjacent PAM sequence. Binding of the dCas9:sgRNA complex upstream of the transcription start site interferes with transcription initiation by preventing recruitment of the RNA polymerase while its assembly at a downstream site prevents transcription elongation. ( B ) Schematic representation of the transfection vectors pGTL-1 and pGTL-2. In pGTL-1, a single guide RNA with a 20 nt targeting sequence and the dCas9 protein are coexpressed under Drosophila constitutive promoters U6:3 and Actin5C , respectively. The dCas9 is separated from the blasticidin resistance gene (Bla R ) and eGFP by self-cleaving T2A peptides (dT2A and T2A). The guide RNA (gRNA) scaffold contains the U6 transcription terminator sequence. The pGTL-2 vector contains an additional sgRNA scaffold under the U6:1 promoter, thus allowing production of two sgRNAs simultaneously with the dCas9 protein. The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation.

    Techniques Used: CRISPR, Sequencing, Binding Assay, Transfection, Plasmid Preparation, FLAG-tag

    The roX2 transcript is efficiently down-regulated by CRISPRi. ( A ) Schematic representation of the Drosophila roX2 locus showing relative positions of the roX2 targeting sgRNAs. The transcription start site is marked by an arrow while the intron within the roX2 gene is shown by a solid grey line. The intergenic region between the roX2 transcription start site and the adjacent gene ( cg11695 ) is shown in solid black line. The sgRNAs targeting the rT and rNT strands are shown in red and violet, respectively. The arrowheads mark the position of the primers used to detect roX2 RNA for RT-qPCR analysis. ( B and C ) RT-qPCR measurement of abundance of the roX1 and roX2 transcripts in cell lines coexpressing human (B) or Drosophila (C) dCas9 protein and two sgRNAs (as shown on the x-axis). While sgRNAs rTb+rTc do not affect roX1 or roX2 levels in presence of human dCas9, they specifically knockdown roX2 transcript levels in cells coexpressing Drosophila dCas9. The y-axis shows the enrichment of RNAs relative to rp49 transcript and normalized to the cells transfected with pGTL-1 containing a non-targeting sequence (Ctrl). The data are shown from two biological replicates, each performed in triplicate. The error bars indicate SEM. The % shows knockdown in percentage as compared with the Ctrl.
    Figure Legend Snippet: The roX2 transcript is efficiently down-regulated by CRISPRi. ( A ) Schematic representation of the Drosophila roX2 locus showing relative positions of the roX2 targeting sgRNAs. The transcription start site is marked by an arrow while the intron within the roX2 gene is shown by a solid grey line. The intergenic region between the roX2 transcription start site and the adjacent gene ( cg11695 ) is shown in solid black line. The sgRNAs targeting the rT and rNT strands are shown in red and violet, respectively. The arrowheads mark the position of the primers used to detect roX2 RNA for RT-qPCR analysis. ( B and C ) RT-qPCR measurement of abundance of the roX1 and roX2 transcripts in cell lines coexpressing human (B) or Drosophila (C) dCas9 protein and two sgRNAs (as shown on the x-axis). While sgRNAs rTb+rTc do not affect roX1 or roX2 levels in presence of human dCas9, they specifically knockdown roX2 transcript levels in cells coexpressing Drosophila dCas9. The y-axis shows the enrichment of RNAs relative to rp49 transcript and normalized to the cells transfected with pGTL-1 containing a non-targeting sequence (Ctrl). The data are shown from two biological replicates, each performed in triplicate. The error bars indicate SEM. The % shows knockdown in percentage as compared with the Ctrl.

    Techniques Used: Quantitative RT-PCR, Transfection, Sequencing

    Knock down of roX1 and roX2 RNAs in vivo . ( A ) Schematic representation of the Drosophila transgenesis vectors pGTL-3. The dominant selection marker mini-white is followed by the dual guide RNA expressing cassette under the control of U6:1 and U6:3 promoters while the expression of Drosophila dCas9 is controlled by the constitutively active promoter Actin5c . The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation. ( B ) RT-qPCR analysis showing robust depletion of roX1 and roX2 RNAs in the males of fly lines expressing the corresponding guide RNAs (shown on the x-axis). The y-axis shows the enrichment of RNAs relative to rp49 transcript and normalized to the wild-type ( y,w ) males. The data are shown from two biological replicates, each performed in triplicate. The error bars indicate SEM. The % shows knockdown in percentage as compared with the wild-type. ( C ) Table showing a severe reduction in the male progeny simultaneously expressing roX1 and roX2 guide RNAs (rT8+rT9-Act:dCas9/rTb+rTc-Act:dCas9). The percentage male survival is calculated based on the total number of progeny of the same genotype recovered in the same cross. The y,w strain was used as wild-type.
    Figure Legend Snippet: Knock down of roX1 and roX2 RNAs in vivo . ( A ) Schematic representation of the Drosophila transgenesis vectors pGTL-3. The dominant selection marker mini-white is followed by the dual guide RNA expressing cassette under the control of U6:1 and U6:3 promoters while the expression of Drosophila dCas9 is controlled by the constitutively active promoter Actin5c . The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation. ( B ) RT-qPCR analysis showing robust depletion of roX1 and roX2 RNAs in the males of fly lines expressing the corresponding guide RNAs (shown on the x-axis). The y-axis shows the enrichment of RNAs relative to rp49 transcript and normalized to the wild-type ( y,w ) males. The data are shown from two biological replicates, each performed in triplicate. The error bars indicate SEM. The % shows knockdown in percentage as compared with the wild-type. ( C ) Table showing a severe reduction in the male progeny simultaneously expressing roX1 and roX2 guide RNAs (rT8+rT9-Act:dCas9/rTb+rTc-Act:dCas9). The percentage male survival is calculated based on the total number of progeny of the same genotype recovered in the same cross. The y,w strain was used as wild-type.

    Techniques Used: In Vivo, Selection, Marker, Expressing, Sequencing, FLAG-tag, Quantitative RT-PCR, Activated Clotting Time Assay

    6) Product Images from "Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways"

    Article Title: Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25356

    Kinase-independent functions of PAK5 S364L and D421N activate ERK but do not promote proliferation A. Lysates from hMELT stable cell lines, incubated in 2% serum for 18 hours, were analyzed by SDS-PAGE followed by immunoblotting. Bar graphs show phospho-protein levels normalized to total protein and relative to parental hMELT cells. At least four biological replicates were run with the mean and standard deviation indicated. A representative immunoblot is shown below. † = p
    Figure Legend Snippet: Kinase-independent functions of PAK5 S364L and D421N activate ERK but do not promote proliferation A. Lysates from hMELT stable cell lines, incubated in 2% serum for 18 hours, were analyzed by SDS-PAGE followed by immunoblotting. Bar graphs show phospho-protein levels normalized to total protein and relative to parental hMELT cells. At least four biological replicates were run with the mean and standard deviation indicated. A representative immunoblot is shown below. † = p

    Techniques Used: Stable Transfection, Incubation, SDS Page, Standard Deviation

    Melanoma-common PAK5 mutants do not alter kinase activity A. Protein diagram depicting the location of human PAK5 variants examined in this study. Dots are color-coordinated to divide the mutations into groups: the most frequent melanoma variants (E144K, M173I, E294K, S364L and D421N) in blue, a kinase dead mutant (K478/9R, KD) in red, and a constitutive active mutant (S573N, CA) in green. PBD = p21-binding domain, PS/AID = pseudosubstrate/auto-inhibitory domain, Kinase = kinase domain. Orange boxes represent proline-rich motifs (PxxP). B. Immunoblot showing the stable expression of FLAG-tagged PAK5 variants in immortalized human primary melanocytes (hMELTs). ‘hMELT’ indicates the parental cell line. Shown below each lane is the β-ACTIN-normalized level of mutant PAK5 expression relative to PAK5 wildtype (WT). C. PAK5 kinase activity was measured using the ADP-Glo system and normalized to immunoprecipitated protein levels as measured by immunoblot. A representative immunoblot of immunoprecipitated FLAG-PAK5 is shown. Each bar represents three biological replicates with the mean and standard deviation indicated. † = p
    Figure Legend Snippet: Melanoma-common PAK5 mutants do not alter kinase activity A. Protein diagram depicting the location of human PAK5 variants examined in this study. Dots are color-coordinated to divide the mutations into groups: the most frequent melanoma variants (E144K, M173I, E294K, S364L and D421N) in blue, a kinase dead mutant (K478/9R, KD) in red, and a constitutive active mutant (S573N, CA) in green. PBD = p21-binding domain, PS/AID = pseudosubstrate/auto-inhibitory domain, Kinase = kinase domain. Orange boxes represent proline-rich motifs (PxxP). B. Immunoblot showing the stable expression of FLAG-tagged PAK5 variants in immortalized human primary melanocytes (hMELTs). ‘hMELT’ indicates the parental cell line. Shown below each lane is the β-ACTIN-normalized level of mutant PAK5 expression relative to PAK5 wildtype (WT). C. PAK5 kinase activity was measured using the ADP-Glo system and normalized to immunoprecipitated protein levels as measured by immunoblot. A representative immunoblot of immunoprecipitated FLAG-PAK5 is shown. Each bar represents three biological replicates with the mean and standard deviation indicated. † = p

    Techniques Used: Activity Assay, Mutagenesis, Binding Assay, Expressing, Immunoprecipitation, Standard Deviation

    Melanoma-associated PAK5 mutants do not affect in vitro melanocyte migration or resistance to temozolomide A. hMELT stable cell lines were placed in the seeding port of the depicted microfluidics device. These cells were serum-starved while full serum medium was added to the flanking collection ports to stimulate migration. Using time-lapse microscopy, images were taken every five minutes for a total of twelve hours. Shown is a representative image of cell tracking through the microfluidics device channels using ImageJ. B. The average speed (µm/min) at which hMELT stable cell lines traversed the microfluidics device is shown relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). C. The ability of single cells to persistently migrate in one direction is shown for each PAK5 mutant relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). D. hMELT parental and wildtype (WT) PAK5 cells were seeded in a 96-well plate and treated with various concentrations of temozolomide ranging from 0 µM to 1 mM. Treatment media was changed every two days for a total of seven days after which cell viability was measured using resazurin metabolism. The IC 50 was determined using the ‘log(inhibitor) vs. response (three parameters)’ analysis in GraphPad Prism software. E. hMELT-PAK5 stable cell lines were seeded in a 96-well plate and treated with either 250 µM temozolomide or DMSO. The treatment media was changed every two days for a total of seven days. Cell viability was measured using resazurin metabolism and normalized to the DMSO control. ‘hMELT’ indicates the parental cell line. Each bar represents five biological replicates performed in triplicate with the mean and standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%).
    Figure Legend Snippet: Melanoma-associated PAK5 mutants do not affect in vitro melanocyte migration or resistance to temozolomide A. hMELT stable cell lines were placed in the seeding port of the depicted microfluidics device. These cells were serum-starved while full serum medium was added to the flanking collection ports to stimulate migration. Using time-lapse microscopy, images were taken every five minutes for a total of twelve hours. Shown is a representative image of cell tracking through the microfluidics device channels using ImageJ. B. The average speed (µm/min) at which hMELT stable cell lines traversed the microfluidics device is shown relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). C. The ability of single cells to persistently migrate in one direction is shown for each PAK5 mutant relative to the parental line. Each bar represents > 130 analyzed cells across at least three biological replicates with standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%). D. hMELT parental and wildtype (WT) PAK5 cells were seeded in a 96-well plate and treated with various concentrations of temozolomide ranging from 0 µM to 1 mM. Treatment media was changed every two days for a total of seven days after which cell viability was measured using resazurin metabolism. The IC 50 was determined using the ‘log(inhibitor) vs. response (three parameters)’ analysis in GraphPad Prism software. E. hMELT-PAK5 stable cell lines were seeded in a 96-well plate and treated with either 250 µM temozolomide or DMSO. The treatment media was changed every two days for a total of seven days. Cell viability was measured using resazurin metabolism and normalized to the DMSO control. ‘hMELT’ indicates the parental cell line. Each bar represents five biological replicates performed in triplicate with the mean and standard error of the mean indicated. p -values were calculated using multiple t-tests and significance determined by FDR (Q = 5%).

    Techniques Used: In Vitro, Migration, Stable Transfection, Time-lapse Microscopy, Cell Tracking Assay, Mutagenesis, Software

    PAK5 S364L and D421N promote melanocyte proliferation and ERK activation A. hMELT stable cell lines were placed in 2% serum for 18 hours and then labeled with EdU for eight hours. After labeling, cells were fixed for flow cytometric analysis. Shown is the average percentage of EdU positive cells across at least three biological replicates with error bars representing the standard deviation. † = p
    Figure Legend Snippet: PAK5 S364L and D421N promote melanocyte proliferation and ERK activation A. hMELT stable cell lines were placed in 2% serum for 18 hours and then labeled with EdU for eight hours. After labeling, cells were fixed for flow cytometric analysis. Shown is the average percentage of EdU positive cells across at least three biological replicates with error bars representing the standard deviation. † = p

    Techniques Used: Activation Assay, Stable Transfection, Labeling, Flow Cytometry, Standard Deviation

    PAK5 S364L and D421N promote proliferation through PKA activation A. Lysates from hMELT stable cell lines, incubated in 2% serum for 18 hours, were analyzed by SDS-PAGE followed by immunoblotting with an antibody that recognizes the phosphorylated substrate motif of AGC kinases. ‘hMELT’ indicates the parental cell line. Each bar indicates the average, β-ACTIN-normalized, pAGC substrate signal relative to parental hMELT cells across at least three biological replicates. Error bars represent the standard deviation. † = p
    Figure Legend Snippet: PAK5 S364L and D421N promote proliferation through PKA activation A. Lysates from hMELT stable cell lines, incubated in 2% serum for 18 hours, were analyzed by SDS-PAGE followed by immunoblotting with an antibody that recognizes the phosphorylated substrate motif of AGC kinases. ‘hMELT’ indicates the parental cell line. Each bar indicates the average, β-ACTIN-normalized, pAGC substrate signal relative to parental hMELT cells across at least three biological replicates. Error bars represent the standard deviation. † = p

    Techniques Used: Activation Assay, Stable Transfection, Incubation, SDS Page, Standard Deviation

    PAK5 is the most frequently altered PAK family member in melanoma A. Oncoprint generated in cBioPortal depicting alterations in the PAK gene family for 287 melanomas collected by TCGA (provisional data accessed on 08/2016). PAKs are divided into two groups based upon protein sequence homology: group I (PAK1, PAK2 and PAK3) and group II (PAK4, PAK5, and PAK6). B. Protein diagram of human PAK5 depicting the location and frequency of melanoma-associated variants. Green dots represent missense mutations, black dots represent truncating mutations, and purple dots represent splice-site mutations.
    Figure Legend Snippet: PAK5 is the most frequently altered PAK family member in melanoma A. Oncoprint generated in cBioPortal depicting alterations in the PAK gene family for 287 melanomas collected by TCGA (provisional data accessed on 08/2016). PAKs are divided into two groups based upon protein sequence homology: group I (PAK1, PAK2 and PAK3) and group II (PAK4, PAK5, and PAK6). B. Protein diagram of human PAK5 depicting the location and frequency of melanoma-associated variants. Green dots represent missense mutations, black dots represent truncating mutations, and purple dots represent splice-site mutations.

    Techniques Used: Generated, Sequencing

    7) Product Images from "Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells"

    Article Title: Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-019-1427-2

    Induction of PGCLCs from BLIMP1-mkate2 reporter knockin hESCs with a two-step method. a Schematic protocol for PGCLCs induction. b , c FACS analysis of mKate (+) cell induction by aRB27 medium and GK15 medium at day 4 with different pre-induction time; n = 3 independent experiments. d FACS analysis of mKate (+) cell induction at day 4 stimulated by different concentrations of LIF (1 μg/ml or 10 ng/ml) and BMP4 (0, 200, 500 ng/ml); n = 3 independent experiments. e Bright field (left) and fluorescence images (right) of day 4 embryoid bodies (EBs) stimulated by cytokines; scale bar = 200 μm; n = 3 independent experiments. f FACS analysis of mKate and TNAP double-positive cells at day 4; n = 3 independent experiments. g FACS analysis of EpCAM and INTEGRINα6 double-positive cells at day 4; n = 3 independent experiments. h Expression analysis by RT-qPCR for day 4 EBs differentiated form BLIMP1-mkate2 reporter knockin hESCs. Relative expression levels are shown with normalization to hESCs or day 4 EB. n = 3 independent experiments. Data are presented as mean ± SD
    Figure Legend Snippet: Induction of PGCLCs from BLIMP1-mkate2 reporter knockin hESCs with a two-step method. a Schematic protocol for PGCLCs induction. b , c FACS analysis of mKate (+) cell induction by aRB27 medium and GK15 medium at day 4 with different pre-induction time; n = 3 independent experiments. d FACS analysis of mKate (+) cell induction at day 4 stimulated by different concentrations of LIF (1 μg/ml or 10 ng/ml) and BMP4 (0, 200, 500 ng/ml); n = 3 independent experiments. e Bright field (left) and fluorescence images (right) of day 4 embryoid bodies (EBs) stimulated by cytokines; scale bar = 200 μm; n = 3 independent experiments. f FACS analysis of mKate and TNAP double-positive cells at day 4; n = 3 independent experiments. g FACS analysis of EpCAM and INTEGRINα6 double-positive cells at day 4; n = 3 independent experiments. h Expression analysis by RT-qPCR for day 4 EBs differentiated form BLIMP1-mkate2 reporter knockin hESCs. Relative expression levels are shown with normalization to hESCs or day 4 EB. n = 3 independent experiments. Data are presented as mean ± SD

    Techniques Used: Knock-In, FACS, Fluorescence, Expressing, Quantitative RT-PCR

    Generation of BLIMP1-mkate2 reporter knockin hESC lines. a Schematic illustration of the BLIMP1 locus, and the donor construct carrying T2A-mKate2 and hEF1a-Neo-pA fragments. Black boxes indicate the exons. b Screening by PCR of the homologous recombinants for BLIMP1-mkate2 and of the removal of the selection cassettes (loxP-hEF1a-Neo-pA-loxP). The clones bearing BLIMP1-mkate2 were selected for use in the subsequent studies. c A phase-contrast image of the BLIMP1-mkate2 reporter knockin hESCs. Scale bar = 200 μm. d FACS analysis for OCT3/4, SOX2, SSEA4, TRA-1-60, and NANOG expression in reporter knockin hESCs
    Figure Legend Snippet: Generation of BLIMP1-mkate2 reporter knockin hESC lines. a Schematic illustration of the BLIMP1 locus, and the donor construct carrying T2A-mKate2 and hEF1a-Neo-pA fragments. Black boxes indicate the exons. b Screening by PCR of the homologous recombinants for BLIMP1-mkate2 and of the removal of the selection cassettes (loxP-hEF1a-Neo-pA-loxP). The clones bearing BLIMP1-mkate2 were selected for use in the subsequent studies. c A phase-contrast image of the BLIMP1-mkate2 reporter knockin hESCs. Scale bar = 200 μm. d FACS analysis for OCT3/4, SOX2, SSEA4, TRA-1-60, and NANOG expression in reporter knockin hESCs

    Techniques Used: Knock-In, Construct, Polymerase Chain Reaction, Selection, Clone Assay, FACS, Expressing

    8) Product Images from "TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis"

    Article Title: TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201706021

    Polar clearing requires the ability of TPXL-1 to activate Aurora A. (A) Schematics of the protein products of the WT and Aurora A–binding defective (FD) tpxl-1 transgenes. (B) Immunoblots of control (N2) worms and worms expressing TPXL-1 WT or TPXL-1 FD after depletion of endogenous TPXL-1 by RNAi were probed for TPXL-1 and α-tubulin as a loading control. (C) Spindle length calculated by measuring the distance between the centrosomes (Fig. S1 F) is plotted for control (black) and TPXL-1 depleted ( tpxl-1(RNAi) ; gray) embryos and for embryos expressing TPXL-1 WT (green) or TPXL-1 FD (purple) after endogenous TPXL-1 depletion. n = number of embryos. (D) Confocal images of anaphase embryos expressing TPXL-1 WT ::NG ( n = 10) or TPXL-1 FD ::NG ( n = 11) after endogenous TPXL-1 depletion. To visualize TPXL-1::NG on astral microtubules without saturating the aster centers, a gamma of 2.5 was introduced in Photoshop. (E) Time-lapse series of myosin-depleted rga-3/4Δ embryos expressing mKate2::anillin and TPXL-1 WT ( n = 12) or TPXL-1 FD ( n = 8). Embryos were depleted of HCP-4 along with endogenous TPXL-1 to ensure comparable pole separation. (F) Kymographs of the anterior cortex of the embryos in E beginning 180 s after NEBD. (G) Normalized cortical mKate2::anillin fluorescence at the anterior pole; n = number of linescans. (H) Graph plotting the distance between the anterior aster and anterior pole. n = number of embryos. (I) Model illustrating how the activation of Aurora A by TPXL-1 on astral microtubules could generate a diffusible signal that inhibits the accumulation of contractile ring proteins on the polar cortex. All error bars are SEM. Bars, 5 µm.
    Figure Legend Snippet: Polar clearing requires the ability of TPXL-1 to activate Aurora A. (A) Schematics of the protein products of the WT and Aurora A–binding defective (FD) tpxl-1 transgenes. (B) Immunoblots of control (N2) worms and worms expressing TPXL-1 WT or TPXL-1 FD after depletion of endogenous TPXL-1 by RNAi were probed for TPXL-1 and α-tubulin as a loading control. (C) Spindle length calculated by measuring the distance between the centrosomes (Fig. S1 F) is plotted for control (black) and TPXL-1 depleted ( tpxl-1(RNAi) ; gray) embryos and for embryos expressing TPXL-1 WT (green) or TPXL-1 FD (purple) after endogenous TPXL-1 depletion. n = number of embryos. (D) Confocal images of anaphase embryos expressing TPXL-1 WT ::NG ( n = 10) or TPXL-1 FD ::NG ( n = 11) after endogenous TPXL-1 depletion. To visualize TPXL-1::NG on astral microtubules without saturating the aster centers, a gamma of 2.5 was introduced in Photoshop. (E) Time-lapse series of myosin-depleted rga-3/4Δ embryos expressing mKate2::anillin and TPXL-1 WT ( n = 12) or TPXL-1 FD ( n = 8). Embryos were depleted of HCP-4 along with endogenous TPXL-1 to ensure comparable pole separation. (F) Kymographs of the anterior cortex of the embryos in E beginning 180 s after NEBD. (G) Normalized cortical mKate2::anillin fluorescence at the anterior pole; n = number of linescans. (H) Graph plotting the distance between the anterior aster and anterior pole. n = number of embryos. (I) Model illustrating how the activation of Aurora A by TPXL-1 on astral microtubules could generate a diffusible signal that inhibits the accumulation of contractile ring proteins on the polar cortex. All error bars are SEM. Bars, 5 µm.

    Techniques Used: Binding Assay, Western Blot, Expressing, Fluorescence, Activation Assay

    9) Product Images from "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain"

    Article Title: Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3440

    Cre-dependent recovery of AAV capsid sequences from transduced target cells ( a ) An overview of the CREATE selection process. PCR is used to introduce diversity (full visual spectrum vertical band) into a capsid gene fragment (yellow). The fragment is cloned into the rAAV genome harboring the remaining capsid gene (gray) and is used to generate a library of virus variants. The library is injected into Cre transgenic animals and PCR is used to selectively recover capsid sequences from Cre + cells. ( b ) The rAAV-Cap-in-cis-lox rAAV genome. Cre inverts the polyadenylation (pA) sequence flanked by the lox71 and lox66 sites. PCR primers (half arrows) are used to selectively amplify Cre-recombined sequences. ( c ) PCR products from Cre recombination-dependent (top) and -independent (bottom) amplification of capsid library sequences recovered from two Cre + or Cre − mice are shown. Schematics (bottom) show the PCR amplification strategies (see Supplementary Fig. 1 for details). ( d ) Schematic shows the AAV genes within the Rep-AAP AAV helper plasmid and the proteins encoded by the cap gene. Stop codons inserted in the cap gene eliminate VP1, VP2 and VP3 capsid protein expression. ( e ) DNase-resistant AAV vector genomes (vg) produced with the split AAV2/9 rep-AAP and rAAV-Cap-in-cis-lox genome (top) as compared to the vg produced with standard AAV2/9 rep-cap helper and rAAV-UBC-mCherry genome (middle) or with the AAV2/9 rep-AAP and rAAV-UBC-mCherry genome (bottom). N =3 independent trials per group; mean ± s.d.; ** p
    Figure Legend Snippet: Cre-dependent recovery of AAV capsid sequences from transduced target cells ( a ) An overview of the CREATE selection process. PCR is used to introduce diversity (full visual spectrum vertical band) into a capsid gene fragment (yellow). The fragment is cloned into the rAAV genome harboring the remaining capsid gene (gray) and is used to generate a library of virus variants. The library is injected into Cre transgenic animals and PCR is used to selectively recover capsid sequences from Cre + cells. ( b ) The rAAV-Cap-in-cis-lox rAAV genome. Cre inverts the polyadenylation (pA) sequence flanked by the lox71 and lox66 sites. PCR primers (half arrows) are used to selectively amplify Cre-recombined sequences. ( c ) PCR products from Cre recombination-dependent (top) and -independent (bottom) amplification of capsid library sequences recovered from two Cre + or Cre − mice are shown. Schematics (bottom) show the PCR amplification strategies (see Supplementary Fig. 1 for details). ( d ) Schematic shows the AAV genes within the Rep-AAP AAV helper plasmid and the proteins encoded by the cap gene. Stop codons inserted in the cap gene eliminate VP1, VP2 and VP3 capsid protein expression. ( e ) DNase-resistant AAV vector genomes (vg) produced with the split AAV2/9 rep-AAP and rAAV-Cap-in-cis-lox genome (top) as compared to the vg produced with standard AAV2/9 rep-cap helper and rAAV-UBC-mCherry genome (middle) or with the AAV2/9 rep-AAP and rAAV-UBC-mCherry genome (bottom). N =3 independent trials per group; mean ± s.d.; ** p

    Techniques Used: Selection, Polymerase Chain Reaction, Introduce, Clone Assay, Injection, Transgenic Assay, Sequencing, Amplification, Mouse Assay, Plasmid Preparation, Expressing, Produced

    10) Product Images from "Gcn5 and Esa1 function as histone crotonyltransferases to regulate crotonylation-dependent transcription"

    Article Title: Gcn5 and Esa1 function as histone crotonyltransferases to regulate crotonylation-dependent transcription

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.010302

    Crotonate-induced transcription depends on Gcn5 and Esa1. A–H , RT-qPCR analysis of crotonate-treated WT, gcn5 Δ, and Esa1-N-FRB cells. The 2-ΔΔ C t relative quantitative method was used to analyze the level of mRNA expression compared with untreated WT cells. Expression was normalized to ACT1 . Data are represented as the mean fold-change in relative expression due to crotonate addition from at least three independent experiments mean ± S.E.
    Figure Legend Snippet: Crotonate-induced transcription depends on Gcn5 and Esa1. A–H , RT-qPCR analysis of crotonate-treated WT, gcn5 Δ, and Esa1-N-FRB cells. The 2-ΔΔ C t relative quantitative method was used to analyze the level of mRNA expression compared with untreated WT cells. Expression was normalized to ACT1 . Data are represented as the mean fold-change in relative expression due to crotonate addition from at least three independent experiments mean ± S.E.

    Techniques Used: Quantitative RT-PCR, Expressing

    Crotonate affects histone crotonylation in Gcn5- and Esa1-dependent manner. A, WT, acs1 Δ and acs2_DAmP strains were treated with sodium crotonate (10 m m , pH 7.5) for 3.5 h in the presence of 0.8 m sorbitol. Whole cell extracts were immunoblotted with the indicated antibodies. B–F, MS2 spectra of peptides corresponding to a region spanning Lys-9–Arg-17 in histone H3 showing ( B ) unmodified (but deuteroacetylated) K9 and K14 ( green ), ( C ) crotonylated K9 ( red ), and ( D ) crotonylated K14 ( red ). E and F, MS2 spectra of peptides corresponding to a region spanning Lys-18–Arg-21 in histone H3 showing ( E ) unmodified (but deuteroacetylated) K18 and K23 ( green ) and ( F ) crotonylated K23 ( red ). G and H , WT and gcn5 Δ strains were treated with ( G ) increasing concentrations of sodium crotonate (1, 2, 5, and 10 m m , pH 7.5) or sodium acetate (10 m m , pH 7.5) or ( H ) sodium crotonate (10 m m , pH 7.5) for 3.5 h in the presence of 0.8 m sorbitol. Whole cell extracts were immunoblotted with the indicated antibodies. I and J , nontagged (no FRB) and Esa1-N-FRB tagged strains were treated as in G and H in the presence of rapamycin. Whole cell extracts were immunoblotted with the indicated antibodies.
    Figure Legend Snippet: Crotonate affects histone crotonylation in Gcn5- and Esa1-dependent manner. A, WT, acs1 Δ and acs2_DAmP strains were treated with sodium crotonate (10 m m , pH 7.5) for 3.5 h in the presence of 0.8 m sorbitol. Whole cell extracts were immunoblotted with the indicated antibodies. B–F, MS2 spectra of peptides corresponding to a region spanning Lys-9–Arg-17 in histone H3 showing ( B ) unmodified (but deuteroacetylated) K9 and K14 ( green ), ( C ) crotonylated K9 ( red ), and ( D ) crotonylated K14 ( red ). E and F, MS2 spectra of peptides corresponding to a region spanning Lys-18–Arg-21 in histone H3 showing ( E ) unmodified (but deuteroacetylated) K18 and K23 ( green ) and ( F ) crotonylated K23 ( red ). G and H , WT and gcn5 Δ strains were treated with ( G ) increasing concentrations of sodium crotonate (1, 2, 5, and 10 m m , pH 7.5) or sodium acetate (10 m m , pH 7.5) or ( H ) sodium crotonate (10 m m , pH 7.5) for 3.5 h in the presence of 0.8 m sorbitol. Whole cell extracts were immunoblotted with the indicated antibodies. I and J , nontagged (no FRB) and Esa1-N-FRB tagged strains were treated as in G and H in the presence of rapamycin. Whole cell extracts were immunoblotted with the indicated antibodies.

    Techniques Used:

    11) Product Images from "SmartBac, a new baculovirus system for large protein complex production"

    Article Title: SmartBac, a new baculovirus system for large protein complex production

    Journal: Journal of Structural Biology

    doi: 10.1016/j.yjsbx.2019.100003

    Schemes for the expression of large multiprotein complexes. (a) The eight-subunit protein complex to be expressed. The eight genes are divided into two groups according to their sizes. Two long polyproteins are designed with TEV cleavage sites separating the adjacent genes. Represents (b) Schematic representation of Scheme 1 for the expression of multiprotein complexes with a molecular weight less than 600 kDa. Here the acceptor vector 4V1R is used, but 5V1TR can also be used. (c) Schematic representation of Scheme 2 for the expression of multiprotein complexes with a molecular weight greater than 600 kDa. The fluorescent protein in the 4V2G/4V2R donor vector is not expressed because a stop codon has been inserted at the end of the fusion gene, which is located at the upstream of the coding sequence of the fluorescent protein. The coding sequences of EGFP and tagRFP can also be removed by restriction enzyme digestion.
    Figure Legend Snippet: Schemes for the expression of large multiprotein complexes. (a) The eight-subunit protein complex to be expressed. The eight genes are divided into two groups according to their sizes. Two long polyproteins are designed with TEV cleavage sites separating the adjacent genes. Represents (b) Schematic representation of Scheme 1 for the expression of multiprotein complexes with a molecular weight less than 600 kDa. Here the acceptor vector 4V1R is used, but 5V1TR can also be used. (c) Schematic representation of Scheme 2 for the expression of multiprotein complexes with a molecular weight greater than 600 kDa. The fluorescent protein in the 4V2G/4V2R donor vector is not expressed because a stop codon has been inserted at the end of the fusion gene, which is located at the upstream of the coding sequence of the fluorescent protein. The coding sequences of EGFP and tagRFP can also be removed by restriction enzyme digestion.

    Techniques Used: Expressing, Molecular Weight, Plasmid Preparation, Sequencing

    12) Product Images from "SmartBac, a new baculovirus system for large protein complex production"

    Article Title: SmartBac, a new baculovirus system for large protein complex production

    Journal: Journal of Structural Biology

    doi: 10.1016/j.yjsbx.2019.100003

    Examples of multiprotein complexes expressed using the SmartBac system. (a) Fluorescence signals for tagRFP (top) and EGFP (bottom) detected from Sf9 cells transfected with BVE1S547 and BV2863 (see Table 1 ). (b) Coomassie-stained SDS-PAGE gel of human exocyst complex purified using eight different Twin-Strep tagged subunits (BV-SE1 to BV-SE8, see Table 1 ). (c) Coomassie-stained SDS-PAGE gel of human exocyst complex purified from insect cells co-infected with BV-2863 and BV-E1S547 (see Table 1 ). The exocyst complex was purified using Twin-Strep-tagged subunit EXOC5. (d) Electron micrograph of negative-stained recombinant human exocyst complex. The bar represents 100 nm. (e) Representative classes from 2D classification of recombinant human exocyst complex particles. (f) 3D reconstruction of recombinant human exocyst complex based nsEM data. (g) Coomassie-stained SDS-PAGE gel of the human dynactin complex purified by one-step strep-affinity purification. (h) Coomassie-stained 3–8% Native-PAGE gel of purified human dynactin complex after glycerol density gradient centrifugation purification. (i) Single-particle nsEM analysis of recombinant human dynactin complex with the representative raw micrograph (top) and 2D class averages (bottom). Scale bar, 50 nm. (j) Coomassie-stained SDS-PAGE gel of purified recombinant human COPI complex, human dynein complex, human CSN complex and human SCF complex.
    Figure Legend Snippet: Examples of multiprotein complexes expressed using the SmartBac system. (a) Fluorescence signals for tagRFP (top) and EGFP (bottom) detected from Sf9 cells transfected with BVE1S547 and BV2863 (see Table 1 ). (b) Coomassie-stained SDS-PAGE gel of human exocyst complex purified using eight different Twin-Strep tagged subunits (BV-SE1 to BV-SE8, see Table 1 ). (c) Coomassie-stained SDS-PAGE gel of human exocyst complex purified from insect cells co-infected with BV-2863 and BV-E1S547 (see Table 1 ). The exocyst complex was purified using Twin-Strep-tagged subunit EXOC5. (d) Electron micrograph of negative-stained recombinant human exocyst complex. The bar represents 100 nm. (e) Representative classes from 2D classification of recombinant human exocyst complex particles. (f) 3D reconstruction of recombinant human exocyst complex based nsEM data. (g) Coomassie-stained SDS-PAGE gel of the human dynactin complex purified by one-step strep-affinity purification. (h) Coomassie-stained 3–8% Native-PAGE gel of purified human dynactin complex after glycerol density gradient centrifugation purification. (i) Single-particle nsEM analysis of recombinant human dynactin complex with the representative raw micrograph (top) and 2D class averages (bottom). Scale bar, 50 nm. (j) Coomassie-stained SDS-PAGE gel of purified recombinant human COPI complex, human dynein complex, human CSN complex and human SCF complex.

    Techniques Used: Fluorescence, Transfection, Staining, SDS Page, Purification, Infection, Recombinant, Affinity Purification, Clear Native PAGE, Gradient Centrifugation

    13) Product Images from "Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors"

    Article Title: Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.05.006

    Cardiac miRNA-122 Expression Varies between Various Mouse Strains and between Human Individuals (A–C) qPCR analysis of miRNA-122 expression in (A) hearts of female and male mice of FVB strain (n = 5 mice/group), (B) hearts of male mice of various strains (n = 5 of FVB, outbred, C57Bl6/J, DBA, C57BL/6J × FVB, BALB/c mice/group; n = 3 of CBA mice/group), and (C) heart tissues collected from patients suffering from different cardiomyopathies. (D–F) qPCR analysis of (D) HNF1α , (E) HNF3β , and (F) RXRA expression in heart tissues collected from patients suffering from different cardiomyopathies. Bars represent mean ± SEM in all graphs. Sample description: F, female; M, male; number, age of the patient undergoing heart transplantation.
    Figure Legend Snippet: Cardiac miRNA-122 Expression Varies between Various Mouse Strains and between Human Individuals (A–C) qPCR analysis of miRNA-122 expression in (A) hearts of female and male mice of FVB strain (n = 5 mice/group), (B) hearts of male mice of various strains (n = 5 of FVB, outbred, C57Bl6/J, DBA, C57BL/6J × FVB, BALB/c mice/group; n = 3 of CBA mice/group), and (C) heart tissues collected from patients suffering from different cardiomyopathies. (D–F) qPCR analysis of (D) HNF1α , (E) HNF3β , and (F) RXRA expression in heart tissues collected from patients suffering from different cardiomyopathies. Bars represent mean ± SEM in all graphs. Sample description: F, female; M, male; number, age of the patient undergoing heart transplantation.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Crocin Bleaching Assay, Transplantation Assay

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Human iPSC-Derived Cardiomyocytes (A) qPCR analysis of relative miRNA-122 expression in human iPSC-derived cardiomyocytes from three different iPSC donors. (B and C) Flow cytometric analysis of (B) percentage of GFP-expressing cells and (C) median fluorescence intensity (MFI) 96 h after transduction of human iPSC-derived cardiomyocytes with scAAV9-GFP-TS or scAAV9-GFP-iTS vectors. All experiments were performed in duplicate. Bars represent mean ± SD in all graphs.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Human iPSC-Derived Cardiomyocytes (A) qPCR analysis of relative miRNA-122 expression in human iPSC-derived cardiomyocytes from three different iPSC donors. (B and C) Flow cytometric analysis of (B) percentage of GFP-expressing cells and (C) median fluorescence intensity (MFI) 96 h after transduction of human iPSC-derived cardiomyocytes with scAAV9-GFP-TS or scAAV9-GFP-iTS vectors. All experiments were performed in duplicate. Bars represent mean ± SD in all graphs.

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Fluorescence, Transduction

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Positive Control, Isolation, Transfection, Plasmid Preparation

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs (A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 ( HMOX1 ) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 ( HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs (A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 ( HMOX1 ) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 ( HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    14) Product Images from "GoldCLIP: Gel-omitted Ligation-dependent CLIP"

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    Journal: Genomics, Proteomics & Bioinformatics

    doi: 10.1016/j.gpb.2018.04.003

    Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.
    Figure Legend Snippet: Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.

    Techniques Used: Isolation, Labeling, SDS Page, Western Blot, Transfection, Purification, Stable Transfection

    HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.
    Figure Legend Snippet: HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Techniques Used: Flow Cytometry, Stable Transfection, Expressing, Irradiation, Lysis, Magnetic Beads, Purification, Clone Assay, Next-Generation Sequencing, Western Blot, Transfection, Staining, RNA Binding Assay, Cross-linking Immunoprecipitation, Binding Assay

    15) Product Images from "GoldCLIP: Gel-omitted Ligation-dependent CLIP"

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    Journal: Genomics, Proteomics & Bioinformatics

    doi: 10.1016/j.gpb.2018.04.003

    GoldCLIP identified endogenous RNA targets of PTB A. Comparison of genomic distribution of the uniquely-mapped reads identified by Halo-PTB GoldCLIP or the published iCLIP datasets. Color code is indicated in the legend box on the right. TTS, transcription termination site. B. Total number of peaks identified by GoldCLIP. Two different crosslinking conditions (UVC in blue, 254 nm and UVA in orange, 365 nm) are shown with two negative controls: Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). Peaks identified using the published datasets (PTB_iCLIP2) are shown in gray. C. Comparison of Halo-PTB clusters identified by GoldCLIP at the PTBP1 locus. Genomic tracks of reverse transcriptase stops are shown for the different samples, i.e. , Halo-PTB crosslinked with either UVC (PTB_UVC) or UVA (PTB_UVA), iCLIP from endogenous PTB (PTB_iCLIP2), Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). D. and E. show the highly correlated Pearson’s coefficient between the number of reads obtained from two biological replicates in a 500 bp window across the whole genome for UVC crosslinking (D) and UVA (E), respectively. F. Top HOMER motifs calculated from the peak reads after UVC crosslinking are shown. G. Over-represented Halo-PTB binding motifs identified by GoldCLIP after UVC crosslinking. Histogram of Z-scores indicates the enrichment of hexamers in GoldCLIP clusters compared to randomly chosen regions of similar sizes in the same genes. Z-scores of the top three hexamers are indicated. H. Heatmap showing the coverage of Halo-PTB binding motifs at crosslink clusters that are defined with a 3-nt clustering window. The clusters are sorted from the shortest to the longest. The nucleotide preceding the start and the nucleotide following the median end of all clusters are marked by white lines in the plot. A color key for the coverage per nucleotide of the PTB-binding motifs is shown on the right. I. Similar to F except UVA crosslinking condition was used. J. Similar to G except UVA crosslinking condition was used. K. Similar to H except UVA crosslinking condition was used.
    Figure Legend Snippet: GoldCLIP identified endogenous RNA targets of PTB A. Comparison of genomic distribution of the uniquely-mapped reads identified by Halo-PTB GoldCLIP or the published iCLIP datasets. Color code is indicated in the legend box on the right. TTS, transcription termination site. B. Total number of peaks identified by GoldCLIP. Two different crosslinking conditions (UVC in blue, 254 nm and UVA in orange, 365 nm) are shown with two negative controls: Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). Peaks identified using the published datasets (PTB_iCLIP2) are shown in gray. C. Comparison of Halo-PTB clusters identified by GoldCLIP at the PTBP1 locus. Genomic tracks of reverse transcriptase stops are shown for the different samples, i.e. , Halo-PTB crosslinked with either UVC (PTB_UVC) or UVA (PTB_UVA), iCLIP from endogenous PTB (PTB_iCLIP2), Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). D. and E. show the highly correlated Pearson’s coefficient between the number of reads obtained from two biological replicates in a 500 bp window across the whole genome for UVC crosslinking (D) and UVA (E), respectively. F. Top HOMER motifs calculated from the peak reads after UVC crosslinking are shown. G. Over-represented Halo-PTB binding motifs identified by GoldCLIP after UVC crosslinking. Histogram of Z-scores indicates the enrichment of hexamers in GoldCLIP clusters compared to randomly chosen regions of similar sizes in the same genes. Z-scores of the top three hexamers are indicated. H. Heatmap showing the coverage of Halo-PTB binding motifs at crosslink clusters that are defined with a 3-nt clustering window. The clusters are sorted from the shortest to the longest. The nucleotide preceding the start and the nucleotide following the median end of all clusters are marked by white lines in the plot. A color key for the coverage per nucleotide of the PTB-binding motifs is shown on the right. I. Similar to F except UVA crosslinking condition was used. J. Similar to G except UVA crosslinking condition was used. K. Similar to H except UVA crosslinking condition was used.

    Techniques Used: Binding Assay

    16) Product Images from "BioID identifies proteins involved in the cell biology of caveolae"

    Article Title: BioID identifies proteins involved in the cell biology of caveolae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209856

    CD2AP co-precipitates specifically with cavin1 and caveolin1. A. Western blots labelled with anti-cavin1 antibodies to show input lysates from cells transfected with cavin1-mCherry and constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were solubilised in 0.1% TritonX100 and lysates cleared of insoluble material by centrifugation at 100,000g. Note that even low concentrations of detergent separate complexes of cavin and caveolin proteins. B. Western blots labelled with anti-caveolin1 antibodies to show input lysates from cells transfected with the constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were cross-linked with 0.5mM DSP prior to solubilisation in 1% octylglucoside, 1% TritonX100. Each transfection and precipitation was carried out in duplicate.
    Figure Legend Snippet: CD2AP co-precipitates specifically with cavin1 and caveolin1. A. Western blots labelled with anti-cavin1 antibodies to show input lysates from cells transfected with cavin1-mCherry and constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were solubilised in 0.1% TritonX100 and lysates cleared of insoluble material by centrifugation at 100,000g. Note that even low concentrations of detergent separate complexes of cavin and caveolin proteins. B. Western blots labelled with anti-caveolin1 antibodies to show input lysates from cells transfected with the constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were cross-linked with 0.5mM DSP prior to solubilisation in 1% octylglucoside, 1% TritonX100. Each transfection and precipitation was carried out in duplicate.

    Techniques Used: Western Blot, Transfection, Construct, Immunoprecipitation, Centrifugation

    CSDE1 controls expression of components of caveolae. A . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by Western blotting with the antibodies shown. Three different single siRNA species were used, as well as a pooled population. B . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by indirect immunofluorescence. Two different single siRNA species were used. Cell nucleii are stained with propidium iodide. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. C . SiRNAs against CSDE1 reduce levels of multiple caveolar components, as judged by Western blotting with the antibodies shown. A pooled population of siRNAs was used. D . SiRNAs against CSDE1 cause delocalisation of EHD2, consistent with loss of recruitment to caveolae. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. E . Quantitative PCR to measure changes in cavin1, caveolin1 and CSDE1 mRNA levels relative to mock-transfected cells. Cells were transfected with the pooled siRNAs shown, or with plasmid for transient over-expression of CSDE1. Bars are SD, N = 3. The experiment was repeated twice with equivalent results.
    Figure Legend Snippet: CSDE1 controls expression of components of caveolae. A . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by Western blotting with the antibodies shown. Three different single siRNA species were used, as well as a pooled population. B . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by indirect immunofluorescence. Two different single siRNA species were used. Cell nucleii are stained with propidium iodide. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. C . SiRNAs against CSDE1 reduce levels of multiple caveolar components, as judged by Western blotting with the antibodies shown. A pooled population of siRNAs was used. D . SiRNAs against CSDE1 cause delocalisation of EHD2, consistent with loss of recruitment to caveolae. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. E . Quantitative PCR to measure changes in cavin1, caveolin1 and CSDE1 mRNA levels relative to mock-transfected cells. Cells were transfected with the pooled siRNAs shown, or with plasmid for transient over-expression of CSDE1. Bars are SD, N = 3. The experiment was repeated twice with equivalent results.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression

    Candidate cavin1-interacting proteins identified by BioID. To show all proteins from 7 pooled BioID experiments with a normalised product of enrichment scores greater than that of caveolin1 –the known caveolar protein to which the scores were normalised. A score of zero means that the specified protein was not detected in that experiment. Protein names are colour coded: green–known caveolar component, grey–nuclear protein. To aid visualisation, enrichment scores are shaded with a higher score having stronger shading.
    Figure Legend Snippet: Candidate cavin1-interacting proteins identified by BioID. To show all proteins from 7 pooled BioID experiments with a normalised product of enrichment scores greater than that of caveolin1 –the known caveolar protein to which the scores were normalised. A score of zero means that the specified protein was not detected in that experiment. Protein names are colour coded: green–known caveolar component, grey–nuclear protein. To aid visualisation, enrichment scores are shaded with a higher score having stronger shading.

    Techniques Used:

    Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. Streptavidin reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with streptavidin-HRP. Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.
    Figure Legend Snippet: Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. Streptavidin reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with streptavidin-HRP. Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.

    Techniques Used: Expressing, Construct, Western Blot, Transfection, Staining, Incubation

    17) Product Images from "Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells"

    Article Title: Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-019-1427-2

    Generation of BLIMP1-mkate2 reporter knockin hESC lines. a Schematic illustration of the BLIMP1 locus, and the donor construct carrying T2A-mKate2 and hEF1a-Neo-pA fragments. Black boxes indicate the exons. b Screening by PCR of the homologous recombinants for BLIMP1-mkate2 and of the removal of the selection cassettes (loxP-hEF1a-Neo-pA-loxP). The clones bearing BLIMP1-mkate2 were selected for use in the subsequent studies. c A phase-contrast image of the BLIMP1-mkate2 reporter knockin hESCs. Scale bar = 200 μm. d FACS analysis for OCT3/4, SOX2, SSEA4, TRA-1-60, and NANOG expression in reporter knockin hESCs
    Figure Legend Snippet: Generation of BLIMP1-mkate2 reporter knockin hESC lines. a Schematic illustration of the BLIMP1 locus, and the donor construct carrying T2A-mKate2 and hEF1a-Neo-pA fragments. Black boxes indicate the exons. b Screening by PCR of the homologous recombinants for BLIMP1-mkate2 and of the removal of the selection cassettes (loxP-hEF1a-Neo-pA-loxP). The clones bearing BLIMP1-mkate2 were selected for use in the subsequent studies. c A phase-contrast image of the BLIMP1-mkate2 reporter knockin hESCs. Scale bar = 200 μm. d FACS analysis for OCT3/4, SOX2, SSEA4, TRA-1-60, and NANOG expression in reporter knockin hESCs

    Techniques Used: Knock-In, Construct, Polymerase Chain Reaction, Selection, Clone Assay, FACS, Expressing

    18) Product Images from "Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference"

    Article Title: Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw063

    CRISPRi in Drosophila . ( A ) Diagram showing the CRISPR interference (CRISPRi) system. To repress transcription, the catalytically inactive Cas9 protein (dCas9, green) is targeted either to the template or non-template DNA strand based on the targeting sequence of the sgRNA and an adjacent PAM sequence. Binding of the dCas9:sgRNA complex upstream of the transcription start site interferes with transcription initiation by preventing recruitment of the RNA polymerase while its assembly at a downstream site prevents transcription elongation. ( B ) Schematic representation of the transfection vectors pGTL-1 and pGTL-2. In pGTL-1, a single guide RNA with a 20 nt targeting sequence and the dCas9 protein are coexpressed under Drosophila constitutive promoters U6:3 and Actin5C , respectively. The dCas9 is separated from the blasticidin resistance gene (Bla R ) and eGFP by self-cleaving T2A peptides (dT2A and T2A). The guide RNA (gRNA) scaffold contains the U6 transcription terminator sequence. The pGTL-2 vector contains an additional sgRNA scaffold under the U6:1 promoter, thus allowing production of two sgRNAs simultaneously with the dCas9 protein. The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation.
    Figure Legend Snippet: CRISPRi in Drosophila . ( A ) Diagram showing the CRISPR interference (CRISPRi) system. To repress transcription, the catalytically inactive Cas9 protein (dCas9, green) is targeted either to the template or non-template DNA strand based on the targeting sequence of the sgRNA and an adjacent PAM sequence. Binding of the dCas9:sgRNA complex upstream of the transcription start site interferes with transcription initiation by preventing recruitment of the RNA polymerase while its assembly at a downstream site prevents transcription elongation. ( B ) Schematic representation of the transfection vectors pGTL-1 and pGTL-2. In pGTL-1, a single guide RNA with a 20 nt targeting sequence and the dCas9 protein are coexpressed under Drosophila constitutive promoters U6:3 and Actin5C , respectively. The dCas9 is separated from the blasticidin resistance gene (Bla R ) and eGFP by self-cleaving T2A peptides (dT2A and T2A). The guide RNA (gRNA) scaffold contains the U6 transcription terminator sequence. The pGTL-2 vector contains an additional sgRNA scaffold under the U6:1 promoter, thus allowing production of two sgRNAs simultaneously with the dCas9 protein. The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation.

    Techniques Used: CRISPR, Sequencing, Binding Assay, Transfection, Plasmid Preparation, FLAG-tag

    19) Product Images from "Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *"

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.641480

    Use of immunofluorescence microscopy to detect tāpirin proteins displayed on the cell wall of yeast. White light and epifluorescent images for S. cerevisiae EBY100 treated with anti-Calkro_0844 ( A and E ) or anti-Calkro_0845 ( B and F ) antibodies. S. cerevisiae EBY100 expressing Calkro_0844 was observed under white light ( C ) and epifluorescence ( G ) after incubation with anti-Calkro_0844 antibodies. S. cerevisiae EBY100 expressing Calkro_0845 was observed under white light ( D ) and epifluorescence ( H ) after incubation with anti-Calkro_0845 antibodies. Goat anti-rabbit conjugated with DyLight488 was used as a secondary antibody. All images were captured at ×40; scale bar in each image is 50 μm.
    Figure Legend Snippet: Use of immunofluorescence microscopy to detect tāpirin proteins displayed on the cell wall of yeast. White light and epifluorescent images for S. cerevisiae EBY100 treated with anti-Calkro_0844 ( A and E ) or anti-Calkro_0845 ( B and F ) antibodies. S. cerevisiae EBY100 expressing Calkro_0844 was observed under white light ( C ) and epifluorescence ( G ) after incubation with anti-Calkro_0844 antibodies. S. cerevisiae EBY100 expressing Calkro_0845 was observed under white light ( D ) and epifluorescence ( H ) after incubation with anti-Calkro_0845 antibodies. Goat anti-rabbit conjugated with DyLight488 was used as a secondary antibody. All images were captured at ×40; scale bar in each image is 50 μm.

    Techniques Used: Immunofluorescence, Microscopy, Expressing, Incubation

    SDS-PAGE analysis of tāpirin binding to various plant cell wall components and plant biomass. Tāpirins tested include Csac_1073 (class 1) ( A ), Calkro_0844 (class 1) ( B ), and Calkro_0845 (class 2) ( C ), and thermolysin-digested Calkro_0844 (Calkro_0844_ C ) ( D ). Abbreviations for plant biomass substrates are as follows: aSWG, dilute acid-pretreated switchgrass; aPTD, dilute acid-pretreated P. deltoides × P. trichocarpa ; PTD, P. deltoides × P. trichocarpa. B, bound protein liberated from the substrate after boiling in 1× Laemmli buffer; U, free protein. 40 μg of protein was used in all conditions tested; image is representative of three replicates.
    Figure Legend Snippet: SDS-PAGE analysis of tāpirin binding to various plant cell wall components and plant biomass. Tāpirins tested include Csac_1073 (class 1) ( A ), Calkro_0844 (class 1) ( B ), and Calkro_0845 (class 2) ( C ), and thermolysin-digested Calkro_0844 (Calkro_0844_ C ) ( D ). Abbreviations for plant biomass substrates are as follows: aSWG, dilute acid-pretreated switchgrass; aPTD, dilute acid-pretreated P. deltoides × P. trichocarpa ; PTD, P. deltoides × P. trichocarpa. B, bound protein liberated from the substrate after boiling in 1× Laemmli buffer; U, free protein. 40 μg of protein was used in all conditions tested; image is representative of three replicates.

    Techniques Used: SDS Page, Binding Assay

    Crystal structure of thermolysin-digested Calkro_0844_ C . A, schematic representation in spectrum colors from blue on the N terminus to red on the C terminus. A single magnesium ion is depicted as a green sphere . Four α-helices are marked as well as first and last residues of the protective loop. B, cartoon representation rotated 90° to illustrate the triangular shape of the β-helix core as well as two exposed and one protected surfaces. C, view from the top onto hydrophobic surface of the β-helix core (semi-transparent surface representation, CPK colors), protective loop (semi-transparent surface, cyan ), and N and C termini (cartoon, blue and red , respectively). The first and last residues of the protective loop are marked. D, view from the top onto hydrophobic surface of the β-helix core with protective loop, N and C termini removed. Exposed aromatic residues are highlighted in green and are labeled.
    Figure Legend Snippet: Crystal structure of thermolysin-digested Calkro_0844_ C . A, schematic representation in spectrum colors from blue on the N terminus to red on the C terminus. A single magnesium ion is depicted as a green sphere . Four α-helices are marked as well as first and last residues of the protective loop. B, cartoon representation rotated 90° to illustrate the triangular shape of the β-helix core as well as two exposed and one protected surfaces. C, view from the top onto hydrophobic surface of the β-helix core (semi-transparent surface representation, CPK colors), protective loop (semi-transparent surface, cyan ), and N and C termini (cartoon, blue and red , respectively). The first and last residues of the protective loop are marked. D, view from the top onto hydrophobic surface of the β-helix core with protective loop, N and C termini removed. Exposed aromatic residues are highlighted in green and are labeled.

    Techniques Used: Labeling

    20) Product Images from "Automated 3D light-sheet screening with high spatiotemporal resolution reveals mitotic phenotypes"

    Article Title: Automated 3D light-sheet screening with high spatiotemporal resolution reveals mitotic phenotypes

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.245043

    Phenotype analysis of dCas9-ED targeting RGMA regulatory CpGs in 3D HEK293 spheroids. Example images of HEK293 spheroids stably expressing dCas9-ED or dCas9, after transfection with sgRNA designed to epigenetically alter RGMA expression, in the following combinations: (A) dCas9-DNMT3a with sgRNA targeting anti-correlated CpG (cyan), (B) dCas9-TET1 with sgRNA targeting correlated CpG (magenta), (C) dCas9-DNMT3a with sgRNA targeting the TSS (orange), (D) dCas9 with sgRNA targeting anti-correlated CpG (cyan), (E) dCas9 with sgRNA targeting correlated CpG (magenta) and (F) dCas9 with sgRNA targeting the TSS (orange). (G) Summary of phenotypic effects of epigenetic targeting of RGMA expression in HEK293 spheroids, showing the percentage of spheroids displaying abnormal cellular and global properties, including macronuclei formation, extended mitosis duration, reduced spheroid growth and apoptotic condensed DNA (ACD). Scale bar: 50 µm.
    Figure Legend Snippet: Phenotype analysis of dCas9-ED targeting RGMA regulatory CpGs in 3D HEK293 spheroids. Example images of HEK293 spheroids stably expressing dCas9-ED or dCas9, after transfection with sgRNA designed to epigenetically alter RGMA expression, in the following combinations: (A) dCas9-DNMT3a with sgRNA targeting anti-correlated CpG (cyan), (B) dCas9-TET1 with sgRNA targeting correlated CpG (magenta), (C) dCas9-DNMT3a with sgRNA targeting the TSS (orange), (D) dCas9 with sgRNA targeting anti-correlated CpG (cyan), (E) dCas9 with sgRNA targeting correlated CpG (magenta) and (F) dCas9 with sgRNA targeting the TSS (orange). (G) Summary of phenotypic effects of epigenetic targeting of RGMA expression in HEK293 spheroids, showing the percentage of spheroids displaying abnormal cellular and global properties, including macronuclei formation, extended mitosis duration, reduced spheroid growth and apoptotic condensed DNA (ACD). Scale bar: 50 µm.

    Techniques Used: Stable Transfection, Expressing, Transfection

    21) Product Images from "The C. elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion"

    Article Title: The C. elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion

    Journal: bioRxiv

    doi: 10.1101/802355

    egl-43 acts independently of nhr-67 and hda-1 . (A) Expression of GFP::EGL-43L after control and nhr-67 RNAi. The left panels depict Nomarski (DIC) images and the middle panels GFP::EGL-43L expression together with the LAM-1::GFP BM marker at the early-L3 (Pn.p) stage. The right panels show the GFP signals merged with the ACs labelled by the cdh-3 > mCherry::PH reporter in magenta. (B) Quantification of GFP::EGL-43L levels in the AC. (C) Expression of NHR-67::GFP after control and egl-43 RNAi. The left panels depict Nomarski (DIC) images and the middle panels NHR-67::GFP expression together with the LAM-1::GFP BM marker at the early-L3 (Pn.p) stage. The right panels show the GFP signals merged with the ACs labelled by the lin-3 ACEL > mCherry reporter in magenta. (D) Quantification of NHR-67::GFP levels in the AC. (E) Expression of HDA-1::RFP after control and egl-43 RNAi. The left panels depict Nomarski (DIC) images and the middle panels HDA-1::RFP expression in magenta at the mid-L3 stage. The right panels show the RFP signal merged with the ACs labelled by the cdh-3 > GFP marker together with LAM-1::GFP in green. (F) Quantification of HDA-1::RFP levels in the AC. (G) AC-specific expression of cki-1 from the cdh-3 enhancer/promoter in control, egl-43 and nhr-67 RNAi-treated mid-L3 larvae. The left panels depict Nomarski (DIC) images, the middle panels CKI-1::SL2::mNG expression in green and the right panels the ACs labelled by the cdh-3 > mCherry::PH reporter in magenta. (H) Quantification of the AC invasion and (I) proliferation phenotypes in RNAi-treated animals expressing CKI-1::SL2::mNG (gray bars) compared to their control siblings lacking the cdh-3 > cki-1::SL2::mNG transgene (black bars/boxes). The error bars in the box plots in (B) , (D) , (F) and (I) indicate the Min-to-Max values, and the error bars in the bar chart in (H) indicate the standard deviation. Statistical significance was determined with a Student’s t-test and is indicated with n.s. for p > 0.05, * for p
    Figure Legend Snippet: egl-43 acts independently of nhr-67 and hda-1 . (A) Expression of GFP::EGL-43L after control and nhr-67 RNAi. The left panels depict Nomarski (DIC) images and the middle panels GFP::EGL-43L expression together with the LAM-1::GFP BM marker at the early-L3 (Pn.p) stage. The right panels show the GFP signals merged with the ACs labelled by the cdh-3 > mCherry::PH reporter in magenta. (B) Quantification of GFP::EGL-43L levels in the AC. (C) Expression of NHR-67::GFP after control and egl-43 RNAi. The left panels depict Nomarski (DIC) images and the middle panels NHR-67::GFP expression together with the LAM-1::GFP BM marker at the early-L3 (Pn.p) stage. The right panels show the GFP signals merged with the ACs labelled by the lin-3 ACEL > mCherry reporter in magenta. (D) Quantification of NHR-67::GFP levels in the AC. (E) Expression of HDA-1::RFP after control and egl-43 RNAi. The left panels depict Nomarski (DIC) images and the middle panels HDA-1::RFP expression in magenta at the mid-L3 stage. The right panels show the RFP signal merged with the ACs labelled by the cdh-3 > GFP marker together with LAM-1::GFP in green. (F) Quantification of HDA-1::RFP levels in the AC. (G) AC-specific expression of cki-1 from the cdh-3 enhancer/promoter in control, egl-43 and nhr-67 RNAi-treated mid-L3 larvae. The left panels depict Nomarski (DIC) images, the middle panels CKI-1::SL2::mNG expression in green and the right panels the ACs labelled by the cdh-3 > mCherry::PH reporter in magenta. (H) Quantification of the AC invasion and (I) proliferation phenotypes in RNAi-treated animals expressing CKI-1::SL2::mNG (gray bars) compared to their control siblings lacking the cdh-3 > cki-1::SL2::mNG transgene (black bars/boxes). The error bars in the box plots in (B) , (D) , (F) and (I) indicate the Min-to-Max values, and the error bars in the bar chart in (H) indicate the standard deviation. Statistical significance was determined with a Student’s t-test and is indicated with n.s. for p > 0.05, * for p

    Techniques Used: Helicase-dependent Amplification, Expressing, Laser Capture Microdissection, Marker, Standard Deviation

    22) Product Images from "Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference"

    Article Title: Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw063

    CRISPRi in Drosophila . ( A ) Diagram showing the CRISPR interference (CRISPRi) system. To repress transcription, the catalytically inactive Cas9 protein (dCas9, green) is targeted either to the template or non-template DNA strand based on the targeting sequence of the sgRNA and an adjacent PAM sequence. Binding of the dCas9:sgRNA complex upstream of the transcription start site interferes with transcription initiation by preventing recruitment of the RNA polymerase while its assembly at a downstream site prevents transcription elongation. ( B ) Schematic representation of the transfection vectors pGTL-1 and pGTL-2. In pGTL-1, a single guide RNA with a 20 nt targeting sequence and the dCas9 protein are coexpressed under Drosophila constitutive promoters U6:3 and Actin5C , respectively. The dCas9 is separated from the blasticidin resistance gene (Bla R ) and eGFP by self-cleaving T2A peptides (dT2A and T2A). The guide RNA (gRNA) scaffold contains the U6 transcription terminator sequence. The pGTL-2 vector contains an additional sgRNA scaffold under the U6:1 promoter, thus allowing production of two sgRNAs simultaneously with the dCas9 protein. The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation.
    Figure Legend Snippet: CRISPRi in Drosophila . ( A ) Diagram showing the CRISPR interference (CRISPRi) system. To repress transcription, the catalytically inactive Cas9 protein (dCas9, green) is targeted either to the template or non-template DNA strand based on the targeting sequence of the sgRNA and an adjacent PAM sequence. Binding of the dCas9:sgRNA complex upstream of the transcription start site interferes with transcription initiation by preventing recruitment of the RNA polymerase while its assembly at a downstream site prevents transcription elongation. ( B ) Schematic representation of the transfection vectors pGTL-1 and pGTL-2. In pGTL-1, a single guide RNA with a 20 nt targeting sequence and the dCas9 protein are coexpressed under Drosophila constitutive promoters U6:3 and Actin5C , respectively. The dCas9 is separated from the blasticidin resistance gene (Bla R ) and eGFP by self-cleaving T2A peptides (dT2A and T2A). The guide RNA (gRNA) scaffold contains the U6 transcription terminator sequence. The pGTL-2 vector contains an additional sgRNA scaffold under the U6:1 promoter, thus allowing production of two sgRNAs simultaneously with the dCas9 protein. The mutated amino acid residues (D10 > A and H841 > A) in dCas9 are marked with red asterisks (*). N = NLS sequence, F = FLAG epitope, pA = polyadenylation.

    Techniques Used: CRISPR, Sequencing, Binding Assay, Transfection, Plasmid Preparation, FLAG-tag

    23) Product Images from "Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors"

    Article Title: Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.05.006

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Human iPSC-Derived Cardiomyocytes (A) qPCR analysis of relative miRNA-122 expression in human iPSC-derived cardiomyocytes from three different iPSC donors. (B and C) Flow cytometric analysis of (B) percentage of GFP-expressing cells and (C) median fluorescence intensity (MFI) 96 h after transduction of human iPSC-derived cardiomyocytes with scAAV9-GFP-TS or scAAV9-GFP-iTS vectors. All experiments were performed in duplicate. Bars represent mean ± SD in all graphs.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Human iPSC-Derived Cardiomyocytes (A) qPCR analysis of relative miRNA-122 expression in human iPSC-derived cardiomyocytes from three different iPSC donors. (B and C) Flow cytometric analysis of (B) percentage of GFP-expressing cells and (C) median fluorescence intensity (MFI) 96 h after transduction of human iPSC-derived cardiomyocytes with scAAV9-GFP-TS or scAAV9-GFP-iTS vectors. All experiments were performed in duplicate. Bars represent mean ± SD in all graphs.

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Fluorescence, Transduction

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Positive Control, Isolation, Transfection, Plasmid Preparation

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs (A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 ( HMOX1 ) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 ( HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs (A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 ( HMOX1 ) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 ( HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    24) Product Images from "CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3"

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3

    Journal: EvoDevo

    doi: 10.1186/s13227-017-0073-y

    CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif
    Figure Legend Snippet: CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

    Techniques Used: CRISPR, Plasmid Preparation, Modification, Polymerase Chain Reaction, Sequencing

    25) Product Images from "Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae"

    Article Title: Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae

    Journal: BMC Biology

    doi: 10.1186/s12915-019-0718-5

    mRNAs that show reciprocal changes in the translation of uORFs and mORFs at multiple temperatures. a Wiggle track images showing ribosome-protected fragments (RPF) on the GCN4 mRNA in cells cultured at either 20, 30, or 37 °C, in units of rpm (reads per million mapped reads from two replicates at each temperature). The RPF tracks were normalized to the mRNA levels at each temperature to reflect the changes in translation efficiencies (ΔTE) of uORF and mORF as described in the legend to Fig. 2 and the “ Materials and methods ” section. The schematic shows the position of the uORFs (purple rectangles) and mORF (striped pink rectangle). NCC uORFs are shown with striped purple rectangle. AUG uORFs are in purple rectangles. Average change in the TEs of the three NCC uORFs showing significant changes in translation at 20 or 37 °C (Avg. ∆TE NCC uORFs ) is shown. The enlargement of the boxed area is also shown below with start sites of NCC uORFs (bold, underlined) and the − 3 to − 1 and + 4 context nucleotides. The green arrow shows the NCC uORF start site (AUA) that has been previously shown to be used as an upstream start site [ 46 ]. b – g Same as in a but for b the CPA1 mRNA, c ADH4 mRNA, d Dur1,2 mRNA, e ATG40 mRNA, f AGA1 mRNA, g AGA2 mRNA. h , i Wiggle track images of the ALA1 ( h ) and GRS1 ( i ) mRNAs as described in a . The N-terminal extension (striped purple rectangle) and mORF (striped pink rectangle) are shown. Relative TE NTD (TE NTE /TE mORF ratio) reflects the ratio of translation efficiency of initiation at the start site of the NTE (ACG in the case of ALA1 and UUG in the case of GRS1 ) to that of the combined initiation events at NTE and mAUG
    Figure Legend Snippet: mRNAs that show reciprocal changes in the translation of uORFs and mORFs at multiple temperatures. a Wiggle track images showing ribosome-protected fragments (RPF) on the GCN4 mRNA in cells cultured at either 20, 30, or 37 °C, in units of rpm (reads per million mapped reads from two replicates at each temperature). The RPF tracks were normalized to the mRNA levels at each temperature to reflect the changes in translation efficiencies (ΔTE) of uORF and mORF as described in the legend to Fig. 2 and the “ Materials and methods ” section. The schematic shows the position of the uORFs (purple rectangles) and mORF (striped pink rectangle). NCC uORFs are shown with striped purple rectangle. AUG uORFs are in purple rectangles. Average change in the TEs of the three NCC uORFs showing significant changes in translation at 20 or 37 °C (Avg. ∆TE NCC uORFs ) is shown. The enlargement of the boxed area is also shown below with start sites of NCC uORFs (bold, underlined) and the − 3 to − 1 and + 4 context nucleotides. The green arrow shows the NCC uORF start site (AUA) that has been previously shown to be used as an upstream start site [ 46 ]. b – g Same as in a but for b the CPA1 mRNA, c ADH4 mRNA, d Dur1,2 mRNA, e ATG40 mRNA, f AGA1 mRNA, g AGA2 mRNA. h , i Wiggle track images of the ALA1 ( h ) and GRS1 ( i ) mRNAs as described in a . The N-terminal extension (striped purple rectangle) and mORF (striped pink rectangle) are shown. Relative TE NTD (TE NTE /TE mORF ratio) reflects the ratio of translation efficiency of initiation at the start site of the NTE (ACG in the case of ALA1 and UUG in the case of GRS1 ) to that of the combined initiation events at NTE and mAUG

    Techniques Used: Cell Culture

    26) Product Images from "ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes"

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1249

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Figure Legend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Techniques Used: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    27) Product Images from "BioID identifies proteins involved in the cell biology of caveolae"

    Article Title: BioID identifies proteins involved in the cell biology of caveolae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209856

    CD2AP co-precipitates specifically with cavin1 and caveolin1. A. Western blots labelled with anti-cavin1 antibodies to show input lysates from cells transfected with cavin1-mCherry and constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were solubilised in 0.1% TritonX100 and lysates cleared of insoluble material by centrifugation at 100,000g. Note that even low concentrations of detergent separate complexes of cavin and caveolin proteins. B. Western blots labelled with anti-caveolin1 antibodies to show input lysates from cells transfected with the constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were cross-linked with 0.5mM DSP prior to solubilisation in 1% octylglucoside, 1% TritonX100. Each transfection and precipitation was carried out in duplicate.
    Figure Legend Snippet: CD2AP co-precipitates specifically with cavin1 and caveolin1. A. Western blots labelled with anti-cavin1 antibodies to show input lysates from cells transfected with cavin1-mCherry and constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were solubilised in 0.1% TritonX100 and lysates cleared of insoluble material by centrifugation at 100,000g. Note that even low concentrations of detergent separate complexes of cavin and caveolin proteins. B. Western blots labelled with anti-caveolin1 antibodies to show input lysates from cells transfected with the constructs shown for each lane, and eluates after immunoprecipitation with anti-GFP antibodies. Cells were cross-linked with 0.5mM DSP prior to solubilisation in 1% octylglucoside, 1% TritonX100. Each transfection and precipitation was carried out in duplicate.

    Techniques Used: Western Blot, Transfection, Construct, Immunoprecipitation, Centrifugation

    CSDE1 controls expression of components of caveolae. A . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by Western blotting with the antibodies shown. Three different single siRNA species were used, as well as a pooled population. B . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by indirect immunofluorescence. Two different single siRNA species were used. Cell nucleii are stained with propidium iodide. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. C . SiRNAs against CSDE1 reduce levels of multiple caveolar components, as judged by Western blotting with the antibodies shown. A pooled population of siRNAs was used. D . SiRNAs against CSDE1 cause delocalisation of EHD2, consistent with loss of recruitment to caveolae. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. E . Quantitative PCR to measure changes in cavin1, caveolin1 and CSDE1 mRNA levels relative to mock-transfected cells. Cells were transfected with the pooled siRNAs shown, or with plasmid for transient over-expression of CSDE1. Bars are SD, N = 3. The experiment was repeated twice with equivalent results.
    Figure Legend Snippet: CSDE1 controls expression of components of caveolae. A . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by Western blotting with the antibodies shown. Three different single siRNA species were used, as well as a pooled population. B . SiRNAs against CSDE1 reduce caveolin1 levels, as judged by indirect immunofluorescence. Two different single siRNA species were used. Cell nucleii are stained with propidium iodide. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. C . SiRNAs against CSDE1 reduce levels of multiple caveolar components, as judged by Western blotting with the antibodies shown. A pooled population of siRNAs was used. D . SiRNAs against CSDE1 cause delocalisation of EHD2, consistent with loss of recruitment to caveolae. Bars 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. E . Quantitative PCR to measure changes in cavin1, caveolin1 and CSDE1 mRNA levels relative to mock-transfected cells. Cells were transfected with the pooled siRNAs shown, or with plasmid for transient over-expression of CSDE1. Bars are SD, N = 3. The experiment was repeated twice with equivalent results.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression

    Candidate cavin1-interacting proteins identified by BioID. To show all proteins from 7 pooled BioID experiments with a normalised product of enrichment scores greater than that of caveolin1 –the known caveolar protein to which the scores were normalised. A score of zero means that the specified protein was not detected in that experiment. Protein names are colour coded: green–known caveolar component, grey–nuclear protein. To aid visualisation, enrichment scores are shaded with a higher score having stronger shading.
    Figure Legend Snippet: Candidate cavin1-interacting proteins identified by BioID. To show all proteins from 7 pooled BioID experiments with a normalised product of enrichment scores greater than that of caveolin1 –the known caveolar protein to which the scores were normalised. A score of zero means that the specified protein was not detected in that experiment. Protein names are colour coded: green–known caveolar component, grey–nuclear protein. To aid visualisation, enrichment scores are shaded with a higher score having stronger shading.

    Techniques Used:

    Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. Streptavidin reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with streptavidin-HRP. Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.
    Figure Legend Snippet: Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. Streptavidin reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with streptavidin-HRP. Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.

    Techniques Used: Expressing, Construct, Western Blot, Transfection, Staining, Incubation

    28) Product Images from "The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes"

    Article Title: The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000959

    KAP1 (TRIM28) is a major component of SMARCAD1 complexes in mouse ESCs. A , silver-stained SDS-polyacrylamide gel of a representative FLAG-SMARCAD1 and control purification from PGK12.1 ESCs. FLAG-SMARCAD1, KAP1, and M r markers are indicated. B , mass spectrometry data from SMARCAD1 affinity purifications. Uniprot accession numbers are given. The Mascot score ( M ), number of identified unique peptides ( P ) is a semiquantitative analytical value indicating the relative abundance of peptides within the sample. Scores for the corresponding control purification are shown in parentheses. C , Western blot analysis of ESCs biochemically fractionated into chromatin-enriched ( C ) and soluble fractions ( S ). Endogenous SMARCAD1 and KAP1 proteins from E14 cells are readily extractable with 0.1% Triton ( S ); ∼20% of each protein remains associated with the chromatin ( C ). Controls, GAPDH, and histone H3 separate into the appropriate subfractions. D , endogenous SMARCAD1 was immunoprecipitated from J1 ESC extracts in the presence of Benzonase and ethidium bromide. Immunoprecipitates ( IP ) and 5% of the input were separated by SDS-PAGE and KAP1 was detected by immunoblotting as co-immunoprecipitating protein.
    Figure Legend Snippet: KAP1 (TRIM28) is a major component of SMARCAD1 complexes in mouse ESCs. A , silver-stained SDS-polyacrylamide gel of a representative FLAG-SMARCAD1 and control purification from PGK12.1 ESCs. FLAG-SMARCAD1, KAP1, and M r markers are indicated. B , mass spectrometry data from SMARCAD1 affinity purifications. Uniprot accession numbers are given. The Mascot score ( M ), number of identified unique peptides ( P ) is a semiquantitative analytical value indicating the relative abundance of peptides within the sample. Scores for the corresponding control purification are shown in parentheses. C , Western blot analysis of ESCs biochemically fractionated into chromatin-enriched ( C ) and soluble fractions ( S ). Endogenous SMARCAD1 and KAP1 proteins from E14 cells are readily extractable with 0.1% Triton ( S ); ∼20% of each protein remains associated with the chromatin ( C ). Controls, GAPDH, and histone H3 separate into the appropriate subfractions. D , endogenous SMARCAD1 was immunoprecipitated from J1 ESC extracts in the presence of Benzonase and ethidium bromide. Immunoprecipitates ( IP ) and 5% of the input were separated by SDS-PAGE and KAP1 was detected by immunoblotting as co-immunoprecipitating protein.

    Techniques Used: Staining, Purification, Mass Spectrometry, Western Blot, Immunoprecipitation, SDS Page

    SMARCAD1 protein becomes readily extractable upon KAP1 depletion in ESCs. Representative examples of indirect immunofluorescence for KAP1 and SMARCAD1 proteins upon shRNA-mediated knockdown of either SMARCAD1 or KAP1. A and B , stable control knockdown ( Ctrl ) and SMARCAD1 knockdown ( KD ) mouse E14 ESCs were permeabilized, and soluble proteins were extracted before fixation. A , clear knockdown of SMARCAD1. B , KAP1 localization is not affected when SMARCAD1 is depleted. Similar results were obtained when the experiment was repeated in a different ESC line. DNA was counterstained with DAPI. C , KAP1 depletion alters SMARCAD1 binding in the nucleus. Double-staining in PGK12.1 ESCs transfected for 4 days with control or Kap1 shRNA plasmids without antibiotic selection. Conditions chosen allow detection of untransfected and transfected cells in parallel. Two fixation and permeabilization protocols were used. Top , cells were formaldehyde-fixed before permeabilization (not pre-extracted). Bottom , extraction of soluble proteins before fixation (pre-extracted). Dotted circle , example of SMARCAD1 localization in nuclei depleted for KAP1. Scale bar , 10 μm.
    Figure Legend Snippet: SMARCAD1 protein becomes readily extractable upon KAP1 depletion in ESCs. Representative examples of indirect immunofluorescence for KAP1 and SMARCAD1 proteins upon shRNA-mediated knockdown of either SMARCAD1 or KAP1. A and B , stable control knockdown ( Ctrl ) and SMARCAD1 knockdown ( KD ) mouse E14 ESCs were permeabilized, and soluble proteins were extracted before fixation. A , clear knockdown of SMARCAD1. B , KAP1 localization is not affected when SMARCAD1 is depleted. Similar results were obtained when the experiment was repeated in a different ESC line. DNA was counterstained with DAPI. C , KAP1 depletion alters SMARCAD1 binding in the nucleus. Double-staining in PGK12.1 ESCs transfected for 4 days with control or Kap1 shRNA plasmids without antibiotic selection. Conditions chosen allow detection of untransfected and transfected cells in parallel. Two fixation and permeabilization protocols were used. Top , cells were formaldehyde-fixed before permeabilization (not pre-extracted). Bottom , extraction of soluble proteins before fixation (pre-extracted). Dotted circle , example of SMARCAD1 localization in nuclei depleted for KAP1. Scale bar , 10 μm.

    Techniques Used: Immunofluorescence, shRNA, Binding Assay, Double Staining, Transfection, Selection

    The CUE1 domain in SMARCAD1 is important for its interaction with KAP1 in vivo . A , characterization of E14 cells expressing tagged SMARCAD1 constructs. An inducible SMARCAD1 knockdown ESC line ( lane 1 ) shows effective depletion of SMARCAD1 upon 2 days of treatment with doxycycline ( dox ; lane 2 ). The same cell line was stably transfected with FLAG- Smarcad1 -V5 constructs, either wildtype ( WT , lane 3 ) or the CUE1 domain mutant F169K, L196K ( mt , lane 4 ). To avoid possible artifacts that can arise when comparing single cell clones, we chose to work with pools of transfectants. Tagged WT and mutant proteins are expressed to similar levels. KAP1 and pluripotency marker OCT4 levels are not affected by depletion or overexpression of SMARCAD1. Lamin B1 serves as a loading control. B , a FLAG-specific antibody readily co-immunoprecipitates KAP1 from ESCs expressing FLAG-tagged SMARCAD1 WT protein ( lane 3 ). The addition of ethidium bromide and Benzonase demonstrated that the interaction of SMARCAD1 and KAP1 is DNA-independent. KAP1 association with FLAG SMARCAD1 is significantly reduced when the CUE1 domain is mutated at F169K and L196K ( lane 6 ). Lanes 1 and 4 , 3% input; lanes 2 and 5 , IgG. PRMT5 served as a negative control. Dotted line , discontinuous lanes from the same gel. Shown is a co-immunoprecipitation performed under conditions where endogenous SMARCAD1 protein was depleted by 2-day doxycycline treatment. Similar results were observed in independent experiments when SMARCAD1 WT and CUE1 mutant constructs were transiently transfected in ESCs expressing endogenous SMARCAD1. C , SMARCAD1 remodeler harboring the F169K/L196K mutation in the CUE1 domain is readily extractable from ESC nuclei. Co-staining of KAP1 and V5-tagged SMARCAD1 in E14 cells expressing the CUE1 mutant ( CUE1 mt V5 ). ESCs with V5-tagged WT protein served as control ( WT-V5 ). Cells were either stained directly or after extraction of soluble proteins. Similar results were obtained in the presence (not shown) or absence of doxycycline (shown). Scale bar , 10 μm.
    Figure Legend Snippet: The CUE1 domain in SMARCAD1 is important for its interaction with KAP1 in vivo . A , characterization of E14 cells expressing tagged SMARCAD1 constructs. An inducible SMARCAD1 knockdown ESC line ( lane 1 ) shows effective depletion of SMARCAD1 upon 2 days of treatment with doxycycline ( dox ; lane 2 ). The same cell line was stably transfected with FLAG- Smarcad1 -V5 constructs, either wildtype ( WT , lane 3 ) or the CUE1 domain mutant F169K, L196K ( mt , lane 4 ). To avoid possible artifacts that can arise when comparing single cell clones, we chose to work with pools of transfectants. Tagged WT and mutant proteins are expressed to similar levels. KAP1 and pluripotency marker OCT4 levels are not affected by depletion or overexpression of SMARCAD1. Lamin B1 serves as a loading control. B , a FLAG-specific antibody readily co-immunoprecipitates KAP1 from ESCs expressing FLAG-tagged SMARCAD1 WT protein ( lane 3 ). The addition of ethidium bromide and Benzonase demonstrated that the interaction of SMARCAD1 and KAP1 is DNA-independent. KAP1 association with FLAG SMARCAD1 is significantly reduced when the CUE1 domain is mutated at F169K and L196K ( lane 6 ). Lanes 1 and 4 , 3% input; lanes 2 and 5 , IgG. PRMT5 served as a negative control. Dotted line , discontinuous lanes from the same gel. Shown is a co-immunoprecipitation performed under conditions where endogenous SMARCAD1 protein was depleted by 2-day doxycycline treatment. Similar results were observed in independent experiments when SMARCAD1 WT and CUE1 mutant constructs were transiently transfected in ESCs expressing endogenous SMARCAD1. C , SMARCAD1 remodeler harboring the F169K/L196K mutation in the CUE1 domain is readily extractable from ESC nuclei. Co-staining of KAP1 and V5-tagged SMARCAD1 in E14 cells expressing the CUE1 mutant ( CUE1 mt V5 ). ESCs with V5-tagged WT protein served as control ( WT-V5 ). Cells were either stained directly or after extraction of soluble proteins. Similar results were obtained in the presence (not shown) or absence of doxycycline (shown). Scale bar , 10 μm.

    Techniques Used: In Vivo, Expressing, Construct, Stable Transfection, Transfection, Mutagenesis, Clone Assay, Marker, Over Expression, Negative Control, Immunoprecipitation, Staining

    KAP1 depletion affects SMARCAD1 RNA and protein levels in ESCs. A , endogenous and exogenously expressed SMARCAD1 levels fall when KAP1 is depleted by 5-day treatment with a specific shRNA ( KD ) compared with untreated ES cells (−). Left , immunoblot detection of the levels of endogenous SMARCAD1 ( lanes 1 and 2 ) and of V5-tagged SMARCAD1 overexpressed in E14 cells (+ WT ; lanes 3 and 4 ). KAP1 levels are significantly reduced ( top ), and endogenous SMARCAD1 levels fall ( SMARCAD1 panel ) as do levels of the exogenous protein ( V5 panel ). Lamin B1 is used as a loading control. Pluripotency marker NANOG levels are reduced upon KAP1 depletion. Right , quantitative RT-PCR analysis of Smarcad1 and Kap1 in the same samples as to the left . Gene expression was normalized to the average of three housekeeping genes and is presented as mean ± S.D. ( error bars ) of three technical replicates. B , analysis of SMARCAD1, KAP1, and lamin B1 in E14 cells depleted for SMARCAD1. Stable knockdown with an shRNA specific for Smarcad1 in parallel with a non-targeting shRNA ( Ctrl ) effectively reduced the expression of SMARCAD1 but had no detectable impact on KAP1 protein ( left ) or RNA levels ( right ). Similar results were obtained with a different shRNA and ES cell line (data not shown). Data are presented as mean ± S.E. of three technical replicates.
    Figure Legend Snippet: KAP1 depletion affects SMARCAD1 RNA and protein levels in ESCs. A , endogenous and exogenously expressed SMARCAD1 levels fall when KAP1 is depleted by 5-day treatment with a specific shRNA ( KD ) compared with untreated ES cells (−). Left , immunoblot detection of the levels of endogenous SMARCAD1 ( lanes 1 and 2 ) and of V5-tagged SMARCAD1 overexpressed in E14 cells (+ WT ; lanes 3 and 4 ). KAP1 levels are significantly reduced ( top ), and endogenous SMARCAD1 levels fall ( SMARCAD1 panel ) as do levels of the exogenous protein ( V5 panel ). Lamin B1 is used as a loading control. Pluripotency marker NANOG levels are reduced upon KAP1 depletion. Right , quantitative RT-PCR analysis of Smarcad1 and Kap1 in the same samples as to the left . Gene expression was normalized to the average of three housekeeping genes and is presented as mean ± S.D. ( error bars ) of three technical replicates. B , analysis of SMARCAD1, KAP1, and lamin B1 in E14 cells depleted for SMARCAD1. Stable knockdown with an shRNA specific for Smarcad1 in parallel with a non-targeting shRNA ( Ctrl ) effectively reduced the expression of SMARCAD1 but had no detectable impact on KAP1 protein ( left ) or RNA levels ( right ). Similar results were obtained with a different shRNA and ES cell line (data not shown). Data are presented as mean ± S.E. of three technical replicates.

    Techniques Used: shRNA, Marker, Quantitative RT-PCR, Expressing

    29) Product Images from "Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells"

    Article Title: Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-019-1427-2

    Generation of BLIMP1-mkate2 reporter knockin hESC lines. a Schematic illustration of the BLIMP1 locus, and the donor construct carrying T2A-mKate2 and hEF1a-Neo-pA fragments. Black boxes indicate the exons. b Screening by PCR of the homologous recombinants for BLIMP1-mkate2 and of the removal of the selection cassettes (loxP-hEF1a-Neo-pA-loxP). The clones bearing BLIMP1-mkate2 were selected for use in the subsequent studies. c A phase-contrast image of the BLIMP1-mkate2 reporter knockin hESCs. Scale bar = 200 μm. d FACS analysis for OCT3/4, SOX2, SSEA4, TRA-1-60, and NANOG expression in reporter knockin hESCs
    Figure Legend Snippet: Generation of BLIMP1-mkate2 reporter knockin hESC lines. a Schematic illustration of the BLIMP1 locus, and the donor construct carrying T2A-mKate2 and hEF1a-Neo-pA fragments. Black boxes indicate the exons. b Screening by PCR of the homologous recombinants for BLIMP1-mkate2 and of the removal of the selection cassettes (loxP-hEF1a-Neo-pA-loxP). The clones bearing BLIMP1-mkate2 were selected for use in the subsequent studies. c A phase-contrast image of the BLIMP1-mkate2 reporter knockin hESCs. Scale bar = 200 μm. d FACS analysis for OCT3/4, SOX2, SSEA4, TRA-1-60, and NANOG expression in reporter knockin hESCs

    Techniques Used: Knock-In, Construct, Polymerase Chain Reaction, Selection, Clone Assay, FACS, Expressing

    30) Product Images from "Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants"

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190526

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.
    Figure Legend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    31) Product Images from "Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors"

    Article Title: Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.05.006

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Human iPSC-Derived Cardiomyocytes (A) qPCR analysis of relative miRNA-122 expression in human iPSC-derived cardiomyocytes from three different iPSC donors. (B and C) Flow cytometric analysis of (B) percentage of GFP-expressing cells and (C) median fluorescence intensity (MFI) 96 h after transduction of human iPSC-derived cardiomyocytes with scAAV9-GFP-TS or scAAV9-GFP-iTS vectors. All experiments were performed in duplicate. Bars represent mean ± SD in all graphs.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Human iPSC-Derived Cardiomyocytes (A) qPCR analysis of relative miRNA-122 expression in human iPSC-derived cardiomyocytes from three different iPSC donors. (B and C) Flow cytometric analysis of (B) percentage of GFP-expressing cells and (C) median fluorescence intensity (MFI) 96 h after transduction of human iPSC-derived cardiomyocytes with scAAV9-GFP-TS or scAAV9-GFP-iTS vectors. All experiments were performed in duplicate. Bars represent mean ± SD in all graphs.

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Fluorescence, Transduction

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Positive Control, Isolation, Transfection, Plasmid Preparation

    Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs (A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 ( HMOX1 ) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 ( HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.
    Figure Legend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs (A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 ( HMOX1 ) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 ( HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Related Articles

    Polymerase Chain Reaction:

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3
    Article Snippet: .. Construction of robo3TcRobo2/3 donor plasmid The initial robo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611). .. The four fragments were derived from pBluescript (plasmid backbone; primer pair 417–418), the wild-type robo3 genomic locus (5′ and 3′ homology regions; primer pairs 501–502 and 505–506), and the robo3 cDNA (robo3 coding region; primer pair 503–504).

    Clone Assay:

    Article Title: TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis
    Article Snippet: .. Transgene construction Gibson cloning (E2611; NEB) was used to construct transgenes encoding WT and mNeonGreen tagged TPXL-1 (isoform A) in pCFJ350. ..

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *
    Article Snippet: .. Tāpirins (Calkro_0844, Calkro_0845) were cloned into the vector pCTCON , using conventional ligation (Calkro_0845) or Gibson Assembly® master mix (Calkro_0844) (New England Biolabs), according to the manufacturer's directions. .. The cloning strategy excluded signal peptides and/or transmembrane domains; oligonucleotide primers used for cloning are listed in .

    Article Title: Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae
    Article Snippet: .. Generation of C-terminally HA-tagged clones of AGA1 gene Gibson assembly master mix (NEB# E2611) was used to generate C-terminally HA-tagged AGA1 clones [AGA 1-HA (WT), see plasmid #12, Additional file : Table S2]. ..

    Construct:

    Article Title: TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis
    Article Snippet: .. Transgene construction Gibson cloning (E2611; NEB) was used to construct transgenes encoding WT and mNeonGreen tagged TPXL-1 (isoform A) in pCFJ350. ..

    Article Title: A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment
    Article Snippet: .. Constructs were built by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Cat. # E2621). .. Tables listing the PCR template and a pair of oligos used to amplify each DNA fragment used to build plasmids generated in this work can be found in the “Supplementary Methods” section in the Supplementary Information file.

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3
    Article Snippet: .. Construction of robo3TcRobo2/3 donor plasmid The initial robo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611). .. The four fragments were derived from pBluescript (plasmid backbone; primer pair 417–418), the wild-type robo3 genomic locus (5′ and 3′ homology regions; primer pairs 501–502 and 505–506), and the robo3 cDNA (robo3 coding region; primer pair 503–504).

    Plasmid Preparation:

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *
    Article Snippet: .. Tāpirins (Calkro_0844, Calkro_0845) were cloned into the vector pCTCON , using conventional ligation (Calkro_0845) or Gibson Assembly® master mix (Calkro_0844) (New England Biolabs), according to the manufacturer's directions. .. The cloning strategy excluded signal peptides and/or transmembrane domains; oligonucleotide primers used for cloning are listed in .

    Article Title: Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae
    Article Snippet: .. Generation of C-terminally HA-tagged clones of AGA1 gene Gibson assembly master mix (NEB# E2611) was used to generate C-terminally HA-tagged AGA1 clones [AGA 1-HA (WT), see plasmid #12, Additional file : Table S2]. ..

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3
    Article Snippet: .. Construction of robo3TcRobo2/3 donor plasmid The initial robo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611). .. The four fragments were derived from pBluescript (plasmid backbone; primer pair 417–418), the wild-type robo3 genomic locus (5′ and 3′ homology regions; primer pairs 501–502 and 505–506), and the robo3 cDNA (robo3 coding region; primer pair 503–504).

    Ligation:

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *
    Article Snippet: .. Tāpirins (Calkro_0844, Calkro_0845) were cloned into the vector pCTCON , using conventional ligation (Calkro_0845) or Gibson Assembly® master mix (Calkro_0844) (New England Biolabs), according to the manufacturer's directions. .. The cloning strategy excluded signal peptides and/or transmembrane domains; oligonucleotide primers used for cloning are listed in .

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    New England Biolabs gibson assembly master mix
    A large-scale DNA data storage in living cell. a) The workflow for the manufacture of <t>mixed</t> culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to <t>assembly</t> and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original <t>master</t> pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.
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    A large-scale DNA data storage in living cell. a) The workflow for the manufacture of mixed culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to assembly and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original master pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.

    Journal: bioRxiv

    Article Title: Mixed Culture of Bacterial Cell for Large Scale DNA Storage

    doi: 10.1101/2020.02.21.960476

    Figure Lengend Snippet: A large-scale DNA data storage in living cell. a) The workflow for the manufacture of mixed culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to assembly and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original master pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.

    Article Snippet: Then the Gibson Assembly® Master Mix – Assembly (NEB, #E2611) was used according to user’s manual.

    Techniques: Transformation Assay, Amplification, Plasmid Preparation, Passaging, Sequencing

    Polar clearing requires the ability of TPXL-1 to activate Aurora A. (A) Schematics of the protein products of the WT and Aurora A–binding defective (FD) tpxl-1 transgenes. (B) Immunoblots of control (N2) worms and worms expressing TPXL-1 WT or TPXL-1 FD after depletion of endogenous TPXL-1 by RNAi were probed for TPXL-1 and α-tubulin as a loading control. (C) Spindle length calculated by measuring the distance between the centrosomes (Fig. S1 F) is plotted for control (black) and TPXL-1 depleted ( tpxl-1(RNAi) ; gray) embryos and for embryos expressing TPXL-1 WT (green) or TPXL-1 FD (purple) after endogenous TPXL-1 depletion. n = number of embryos. (D) Confocal images of anaphase embryos expressing TPXL-1 WT ::NG ( n = 10) or TPXL-1 FD ::NG ( n = 11) after endogenous TPXL-1 depletion. To visualize TPXL-1::NG on astral microtubules without saturating the aster centers, a gamma of 2.5 was introduced in Photoshop. (E) Time-lapse series of myosin-depleted rga-3/4Δ embryos expressing mKate2::anillin and TPXL-1 WT ( n = 12) or TPXL-1 FD ( n = 8). Embryos were depleted of HCP-4 along with endogenous TPXL-1 to ensure comparable pole separation. (F) Kymographs of the anterior cortex of the embryos in E beginning 180 s after NEBD. (G) Normalized cortical mKate2::anillin fluorescence at the anterior pole; n = number of linescans. (H) Graph plotting the distance between the anterior aster and anterior pole. n = number of embryos. (I) Model illustrating how the activation of Aurora A by TPXL-1 on astral microtubules could generate a diffusible signal that inhibits the accumulation of contractile ring proteins on the polar cortex. All error bars are SEM. Bars, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis

    doi: 10.1083/jcb.201706021

    Figure Lengend Snippet: Polar clearing requires the ability of TPXL-1 to activate Aurora A. (A) Schematics of the protein products of the WT and Aurora A–binding defective (FD) tpxl-1 transgenes. (B) Immunoblots of control (N2) worms and worms expressing TPXL-1 WT or TPXL-1 FD after depletion of endogenous TPXL-1 by RNAi were probed for TPXL-1 and α-tubulin as a loading control. (C) Spindle length calculated by measuring the distance between the centrosomes (Fig. S1 F) is plotted for control (black) and TPXL-1 depleted ( tpxl-1(RNAi) ; gray) embryos and for embryos expressing TPXL-1 WT (green) or TPXL-1 FD (purple) after endogenous TPXL-1 depletion. n = number of embryos. (D) Confocal images of anaphase embryos expressing TPXL-1 WT ::NG ( n = 10) or TPXL-1 FD ::NG ( n = 11) after endogenous TPXL-1 depletion. To visualize TPXL-1::NG on astral microtubules without saturating the aster centers, a gamma of 2.5 was introduced in Photoshop. (E) Time-lapse series of myosin-depleted rga-3/4Δ embryos expressing mKate2::anillin and TPXL-1 WT ( n = 12) or TPXL-1 FD ( n = 8). Embryos were depleted of HCP-4 along with endogenous TPXL-1 to ensure comparable pole separation. (F) Kymographs of the anterior cortex of the embryos in E beginning 180 s after NEBD. (G) Normalized cortical mKate2::anillin fluorescence at the anterior pole; n = number of linescans. (H) Graph plotting the distance between the anterior aster and anterior pole. n = number of embryos. (I) Model illustrating how the activation of Aurora A by TPXL-1 on astral microtubules could generate a diffusible signal that inhibits the accumulation of contractile ring proteins on the polar cortex. All error bars are SEM. Bars, 5 µm.

    Article Snippet: Transgene construction Gibson cloning (E2611; NEB) was used to construct transgenes encoding WT and mNeonGreen tagged TPXL-1 (isoform A) in pCFJ350.

    Techniques: Binding Assay, Western Blot, Expressing, Fluorescence, Activation Assay

    Use of immunofluorescence microscopy to detect tāpirin proteins displayed on the cell wall of yeast. White light and epifluorescent images for S. cerevisiae EBY100 treated with anti-Calkro_0844 ( A and E ) or anti-Calkro_0845 ( B and F ) antibodies. S. cerevisiae EBY100 expressing Calkro_0844 was observed under white light ( C ) and epifluorescence ( G ) after incubation with anti-Calkro_0844 antibodies. S. cerevisiae EBY100 expressing Calkro_0845 was observed under white light ( D ) and epifluorescence ( H ) after incubation with anti-Calkro_0845 antibodies. Goat anti-rabbit conjugated with DyLight488 was used as a secondary antibody. All images were captured at ×40; scale bar in each image is 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *

    doi: 10.1074/jbc.M115.641480

    Figure Lengend Snippet: Use of immunofluorescence microscopy to detect tāpirin proteins displayed on the cell wall of yeast. White light and epifluorescent images for S. cerevisiae EBY100 treated with anti-Calkro_0844 ( A and E ) or anti-Calkro_0845 ( B and F ) antibodies. S. cerevisiae EBY100 expressing Calkro_0844 was observed under white light ( C ) and epifluorescence ( G ) after incubation with anti-Calkro_0844 antibodies. S. cerevisiae EBY100 expressing Calkro_0845 was observed under white light ( D ) and epifluorescence ( H ) after incubation with anti-Calkro_0845 antibodies. Goat anti-rabbit conjugated with DyLight488 was used as a secondary antibody. All images were captured at ×40; scale bar in each image is 50 μm.

    Article Snippet: Tāpirins (Calkro_0844, Calkro_0845) were cloned into the vector pCTCON , using conventional ligation (Calkro_0845) or Gibson Assembly® master mix (Calkro_0844) (New England Biolabs), according to the manufacturer's directions.

    Techniques: Immunofluorescence, Microscopy, Expressing, Incubation

    SDS-PAGE analysis of tāpirin binding to various plant cell wall components and plant biomass. Tāpirins tested include Csac_1073 (class 1) ( A ), Calkro_0844 (class 1) ( B ), and Calkro_0845 (class 2) ( C ), and thermolysin-digested Calkro_0844 (Calkro_0844_ C ) ( D ). Abbreviations for plant biomass substrates are as follows: aSWG, dilute acid-pretreated switchgrass; aPTD, dilute acid-pretreated P. deltoides × P. trichocarpa ; PTD, P. deltoides × P. trichocarpa. B, bound protein liberated from the substrate after boiling in 1× Laemmli buffer; U, free protein. 40 μg of protein was used in all conditions tested; image is representative of three replicates.

    Journal: The Journal of Biological Chemistry

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *

    doi: 10.1074/jbc.M115.641480

    Figure Lengend Snippet: SDS-PAGE analysis of tāpirin binding to various plant cell wall components and plant biomass. Tāpirins tested include Csac_1073 (class 1) ( A ), Calkro_0844 (class 1) ( B ), and Calkro_0845 (class 2) ( C ), and thermolysin-digested Calkro_0844 (Calkro_0844_ C ) ( D ). Abbreviations for plant biomass substrates are as follows: aSWG, dilute acid-pretreated switchgrass; aPTD, dilute acid-pretreated P. deltoides × P. trichocarpa ; PTD, P. deltoides × P. trichocarpa. B, bound protein liberated from the substrate after boiling in 1× Laemmli buffer; U, free protein. 40 μg of protein was used in all conditions tested; image is representative of three replicates.

    Article Snippet: Tāpirins (Calkro_0844, Calkro_0845) were cloned into the vector pCTCON , using conventional ligation (Calkro_0845) or Gibson Assembly® master mix (Calkro_0844) (New England Biolabs), according to the manufacturer's directions.

    Techniques: SDS Page, Binding Assay

    Crystal structure of thermolysin-digested Calkro_0844_ C . A, schematic representation in spectrum colors from blue on the N terminus to red on the C terminus. A single magnesium ion is depicted as a green sphere . Four α-helices are marked as well as first and last residues of the protective loop. B, cartoon representation rotated 90° to illustrate the triangular shape of the β-helix core as well as two exposed and one protected surfaces. C, view from the top onto hydrophobic surface of the β-helix core (semi-transparent surface representation, CPK colors), protective loop (semi-transparent surface, cyan ), and N and C termini (cartoon, blue and red , respectively). The first and last residues of the protective loop are marked. D, view from the top onto hydrophobic surface of the β-helix core with protective loop, N and C termini removed. Exposed aromatic residues are highlighted in green and are labeled.

    Journal: The Journal of Biological Chemistry

    Article Title: Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose *

    doi: 10.1074/jbc.M115.641480

    Figure Lengend Snippet: Crystal structure of thermolysin-digested Calkro_0844_ C . A, schematic representation in spectrum colors from blue on the N terminus to red on the C terminus. A single magnesium ion is depicted as a green sphere . Four α-helices are marked as well as first and last residues of the protective loop. B, cartoon representation rotated 90° to illustrate the triangular shape of the β-helix core as well as two exposed and one protected surfaces. C, view from the top onto hydrophobic surface of the β-helix core (semi-transparent surface representation, CPK colors), protective loop (semi-transparent surface, cyan ), and N and C termini (cartoon, blue and red , respectively). The first and last residues of the protective loop are marked. D, view from the top onto hydrophobic surface of the β-helix core with protective loop, N and C termini removed. Exposed aromatic residues are highlighted in green and are labeled.

    Article Snippet: Tāpirins (Calkro_0844, Calkro_0845) were cloned into the vector pCTCON , using conventional ligation (Calkro_0845) or Gibson Assembly® master mix (Calkro_0844) (New England Biolabs), according to the manufacturer's directions.

    Techniques: Labeling

    CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

    Journal: EvoDevo

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3

    doi: 10.1186/s13227-017-0073-y

    Figure Lengend Snippet: CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

    Article Snippet: Construction of robo3TcRobo2/3 donor plasmid The initial robo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611).

    Techniques: CRISPR, Plasmid Preparation, Modification, Polymerase Chain Reaction, Sequencing