in vitro transcription translation reactions  (Promega)

 
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    Structured Review

    Promega in vitro transcription translation reactions
    Production of CopA(Z) depends on the integrity of the slippery sequence present <t>in</t> the copA gene (A) The slippery sequence CCCAAAG present in the copA gene at the end of the MBD1-coding segment. Slippage of the ribosome to the -1 frame would cause the ribosome to incorporate Gly-70 after Lys-69 and terminate <t>translation</t> at the following stop codon, generating the CopA(Z) protein. (B) Immunoblot of the lysates of E. coli cells carrying either the pCopA plasmid containing the wt copA gene, or pCopA-mSS in which the slippery sequence was mutated to prevent -1 PRF. The CopA (grey arrowhead) and CopA(Z) (black arrowhead) proteins were detected using anti-His 6 -tag antibodies. Proteins were fractionated in a 4-20% SDS gel. The SS-disrupting mutations (which do not change the sequence of the encoded protein) are underlined. (C) In <t>vitro</t> <t>transcription-translation</t> of a DNA template containing the first 94 codons of copA , followed by an engineered stop codon. The 16.5% Tris-Tricine SDS gel shows the [ 35 S]-labeled products corresponding to the complete 94 amino acid-long protein encoded in the 0 frame (grey arrowhead) or the 70 amino acid-long CopA(Z) produced via -1 PRF (black arrowhead). Disruption of SS in the template mSS by two synonymous mutations (shown in panel B) prevents production of CopA(Z) in vitro. .
    In Vitro Transcription Translation Reactions, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Programmed ribosomal frameshifting generates a copper transporter and a copper chaperone from the same gene"

    Article Title: Programmed ribosomal frameshifting generates a copper transporter and a copper chaperone from the same gene

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2016.12.008

    Production of CopA(Z) depends on the integrity of the slippery sequence present in the copA gene (A) The slippery sequence CCCAAAG present in the copA gene at the end of the MBD1-coding segment. Slippage of the ribosome to the -1 frame would cause the ribosome to incorporate Gly-70 after Lys-69 and terminate translation at the following stop codon, generating the CopA(Z) protein. (B) Immunoblot of the lysates of E. coli cells carrying either the pCopA plasmid containing the wt copA gene, or pCopA-mSS in which the slippery sequence was mutated to prevent -1 PRF. The CopA (grey arrowhead) and CopA(Z) (black arrowhead) proteins were detected using anti-His 6 -tag antibodies. Proteins were fractionated in a 4-20% SDS gel. The SS-disrupting mutations (which do not change the sequence of the encoded protein) are underlined. (C) In vitro transcription-translation of a DNA template containing the first 94 codons of copA , followed by an engineered stop codon. The 16.5% Tris-Tricine SDS gel shows the [ 35 S]-labeled products corresponding to the complete 94 amino acid-long protein encoded in the 0 frame (grey arrowhead) or the 70 amino acid-long CopA(Z) produced via -1 PRF (black arrowhead). Disruption of SS in the template mSS by two synonymous mutations (shown in panel B) prevents production of CopA(Z) in vitro. .
    Figure Legend Snippet: Production of CopA(Z) depends on the integrity of the slippery sequence present in the copA gene (A) The slippery sequence CCCAAAG present in the copA gene at the end of the MBD1-coding segment. Slippage of the ribosome to the -1 frame would cause the ribosome to incorporate Gly-70 after Lys-69 and terminate translation at the following stop codon, generating the CopA(Z) protein. (B) Immunoblot of the lysates of E. coli cells carrying either the pCopA plasmid containing the wt copA gene, or pCopA-mSS in which the slippery sequence was mutated to prevent -1 PRF. The CopA (grey arrowhead) and CopA(Z) (black arrowhead) proteins were detected using anti-His 6 -tag antibodies. Proteins were fractionated in a 4-20% SDS gel. The SS-disrupting mutations (which do not change the sequence of the encoded protein) are underlined. (C) In vitro transcription-translation of a DNA template containing the first 94 codons of copA , followed by an engineered stop codon. The 16.5% Tris-Tricine SDS gel shows the [ 35 S]-labeled products corresponding to the complete 94 amino acid-long protein encoded in the 0 frame (grey arrowhead) or the 70 amino acid-long CopA(Z) produced via -1 PRF (black arrowhead). Disruption of SS in the template mSS by two synonymous mutations (shown in panel B) prevents production of CopA(Z) in vitro. .

    Techniques Used: Sequencing, Plasmid Preparation, SDS-Gel, In Vitro, Labeling, Produced

    2) Product Images from "Damaging the Integrated HIV Proviral DNA with TALENs"

    Article Title: Damaging the Integrated HIV Proviral DNA with TALENs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125652

    HT-TALENs and NS-TALENs cleave an HIV-1 DNA fragment in vitro . A. Schematic diagram representing HT-TALENs and NS-TALENs bound to their cognate DNA target sequence (thick lines). Relative locations of the Fok1 endonuclease, Flag epitope tag, and nuclear localization sequence (NLS) are indicated. Asterisks and grey boxes designate where a “NS” coding TALE repeat was used in the 5’ NS-TALEN construction. B. Western blot of in vitro transcription/translation reactions containing no expression plasmids, each TALEN alone, the HT-TALEN pair, or the NS-TALEN pair. C. Gel electrophoresis analysis of in vitro cleavage reactions containing no TALEN plasmids, the HT-TALEN pair, or the NS-TALEN pair. The HIV-1 target DNA fragment size is 747 bp, with expected on-target cleavage products of approximately 430 bp and 317 bp. Quantification of cleavage was performed using ImageJ software and is shown below the gel image. D. The HIV-1 target DNA fragment from (C) was mutated in the 5’ TALE binding site to create a set of triple mutant templates (Mut1-Mut4). The sequences of Mut1-Mut4 are depicted in bold, lowercase font and mutated positions are indicated by asterisks. Cleavage reactions containing either the HT-TALEN or NS-TALEN pairs incubated with the HIV-1 target templates were size fractionated by electrophoresis and quantified by densitometry with ImageJ [ 39 ].
    Figure Legend Snippet: HT-TALENs and NS-TALENs cleave an HIV-1 DNA fragment in vitro . A. Schematic diagram representing HT-TALENs and NS-TALENs bound to their cognate DNA target sequence (thick lines). Relative locations of the Fok1 endonuclease, Flag epitope tag, and nuclear localization sequence (NLS) are indicated. Asterisks and grey boxes designate where a “NS” coding TALE repeat was used in the 5’ NS-TALEN construction. B. Western blot of in vitro transcription/translation reactions containing no expression plasmids, each TALEN alone, the HT-TALEN pair, or the NS-TALEN pair. C. Gel electrophoresis analysis of in vitro cleavage reactions containing no TALEN plasmids, the HT-TALEN pair, or the NS-TALEN pair. The HIV-1 target DNA fragment size is 747 bp, with expected on-target cleavage products of approximately 430 bp and 317 bp. Quantification of cleavage was performed using ImageJ software and is shown below the gel image. D. The HIV-1 target DNA fragment from (C) was mutated in the 5’ TALE binding site to create a set of triple mutant templates (Mut1-Mut4). The sequences of Mut1-Mut4 are depicted in bold, lowercase font and mutated positions are indicated by asterisks. Cleavage reactions containing either the HT-TALEN or NS-TALEN pairs incubated with the HIV-1 target templates were size fractionated by electrophoresis and quantified by densitometry with ImageJ [ 39 ].

    Techniques Used: TALENs, In Vitro, Sequencing, FLAG-tag, Western Blot, Expressing, Nucleic Acid Electrophoresis, Software, Binding Assay, Mutagenesis, Incubation, Electrophoresis

    3) Product Images from "Embryonic stem cell differentiation requires full length Chd1"

    Article Title: Embryonic stem cell differentiation requires full length Chd1

    Journal: Scientific Reports

    doi: 10.1038/srep08007

    N-terminally truncated Chd1 is active in nucleosome assembly but lacks major phosphorylation sites. (a) DNA templates used for in vitro transcription/translation experiments. All sequences contain a Flag-tag at the end of exon 11. a , DNA fragment corresponding to the wt sequence including the natural transcription start site; b , DNA fragment corresponding to the transcript of the exon 2-deleted Chd1 locus; c-h , DNA fragments starting with in-frame ATGs located in downstream exons of the Chd1 gene. The red dot depicts the natural translation start codon. (b) Deletion of exon 2 results in protein translation starting at an ATG in exon 4. 35 S-labeled in vitro translation products were separated by SDS-PAGE and visualized by phosphoimaging. Stars depict Chd1 polypeptides used as size markers; arrows indicate translation products of templates a and b . (c) WT and N-terminal truncation mutants of Chd1 were expressed using the baculovirus system, purified and separated by gel electrophoresis and Coomassie Blue staining. (d) Mouse Chd1 catalyzes the assembly of nucleosomes on relaxed circular DNA in a dose dependent manner. Chromatin assembly reactions were carried out with increasing amounts (5, 10, 20 nM) of recombinant wt Chd1, and DNA supercoiling was analyzed after 70 min. (e) N-terminally truncated Chd1 is capable of efficient nucleosome assembly. Products of nucleosome assembly reactions with wt, Δ1-100 and Δ1-149 proteins (20 nM) were analyzed for DNA supercoiling efficiency. Reference lanes contain topoisomerase I-relaxed ( relaxed ) and supercoiled plasmid DNA ( supercoiled ), respectively. The positions of nicked (N) and fully supercoiled (SC) DNA species are indicated. (f) Chd1 is phosphorylated at the N-terminal SRR. Recombinant wt, Δ1-100 and Δ1-149 proteins were treated with alkaline phosphatase (AP), separated by SDS-PAGE and stained with silver. (g) Mouse Chd1 is a phosphoprotein in vivo . Treatment of protein extracts from Chd1 flox/flox cells with AP caused a mobility shift of Chd1 in SDS-PA gel electrophoresis, which was less pronounced for Chd1ΔSRR extracted from Chd1 Δ2/Δ2 cells. Chd1 was detected by immunoblotting with anti-Chd1 antibodies. Alpha-tubulin was used as a loading control.
    Figure Legend Snippet: N-terminally truncated Chd1 is active in nucleosome assembly but lacks major phosphorylation sites. (a) DNA templates used for in vitro transcription/translation experiments. All sequences contain a Flag-tag at the end of exon 11. a , DNA fragment corresponding to the wt sequence including the natural transcription start site; b , DNA fragment corresponding to the transcript of the exon 2-deleted Chd1 locus; c-h , DNA fragments starting with in-frame ATGs located in downstream exons of the Chd1 gene. The red dot depicts the natural translation start codon. (b) Deletion of exon 2 results in protein translation starting at an ATG in exon 4. 35 S-labeled in vitro translation products were separated by SDS-PAGE and visualized by phosphoimaging. Stars depict Chd1 polypeptides used as size markers; arrows indicate translation products of templates a and b . (c) WT and N-terminal truncation mutants of Chd1 were expressed using the baculovirus system, purified and separated by gel electrophoresis and Coomassie Blue staining. (d) Mouse Chd1 catalyzes the assembly of nucleosomes on relaxed circular DNA in a dose dependent manner. Chromatin assembly reactions were carried out with increasing amounts (5, 10, 20 nM) of recombinant wt Chd1, and DNA supercoiling was analyzed after 70 min. (e) N-terminally truncated Chd1 is capable of efficient nucleosome assembly. Products of nucleosome assembly reactions with wt, Δ1-100 and Δ1-149 proteins (20 nM) were analyzed for DNA supercoiling efficiency. Reference lanes contain topoisomerase I-relaxed ( relaxed ) and supercoiled plasmid DNA ( supercoiled ), respectively. The positions of nicked (N) and fully supercoiled (SC) DNA species are indicated. (f) Chd1 is phosphorylated at the N-terminal SRR. Recombinant wt, Δ1-100 and Δ1-149 proteins were treated with alkaline phosphatase (AP), separated by SDS-PAGE and stained with silver. (g) Mouse Chd1 is a phosphoprotein in vivo . Treatment of protein extracts from Chd1 flox/flox cells with AP caused a mobility shift of Chd1 in SDS-PA gel electrophoresis, which was less pronounced for Chd1ΔSRR extracted from Chd1 Δ2/Δ2 cells. Chd1 was detected by immunoblotting with anti-Chd1 antibodies. Alpha-tubulin was used as a loading control.

    Techniques Used: In Vitro, FLAG-tag, Sequencing, Labeling, SDS Page, Purification, Nucleic Acid Electrophoresis, Staining, Recombinant, Plasmid Preparation, In Vivo, Mobility Shift

    4) Product Images from "Functional Analyses of the Three Simian Hemorrhagic Fever Virus Nonstructural Protein 1 Papain-Like Proteases"

    Article Title: Functional Analyses of the Three Simian Hemorrhagic Fever Virus Nonstructural Protein 1 Papain-Like Proteases

    Journal: Journal of Virology

    doi: 10.1128/JVI.01020-14

    MassSpec analyses of selected polyprotein cleavage products. (A) nsp1α. (B) nsp1α+β. (C) nsp1α+β+γ. Products from in vitro transcription/translation reactions were separated on a 12% NuPAGE bis-tris gel
    Figure Legend Snippet: MassSpec analyses of selected polyprotein cleavage products. (A) nsp1α. (B) nsp1α+β. (C) nsp1α+β+γ. Products from in vitro transcription/translation reactions were separated on a 12% NuPAGE bis-tris gel

    Techniques Used: In Vitro

    5) Product Images from "Theiler's murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism"

    Article Title: Theiler's murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.047571-0

    (a) Schematic representation of the wild-type and mutant TMEV and mengovirus region expressed by in vitro translation. (b) Coupled in vitro transcription–translation. Each plasmid (100 ng) in a 10 µl reaction volume was incubated at 30
    Figure Legend Snippet: (a) Schematic representation of the wild-type and mutant TMEV and mengovirus region expressed by in vitro translation. (b) Coupled in vitro transcription–translation. Each plasmid (100 ng) in a 10 µl reaction volume was incubated at 30

    Techniques Used: Mutagenesis, In Vitro, Plasmid Preparation, Incubation

    (a) 4–20 % SDS-PAGE of TMEV infected (m.o.i. 10) BHK-21 cell extracts from a pulse-labelling experiment. Lysates were prepared 6 h post-infection. (b) In vitro translation reactions of FMDV wild-type and its StopGo mutants.
    Figure Legend Snippet: (a) 4–20 % SDS-PAGE of TMEV infected (m.o.i. 10) BHK-21 cell extracts from a pulse-labelling experiment. Lysates were prepared 6 h post-infection. (b) In vitro translation reactions of FMDV wild-type and its StopGo mutants.

    Techniques Used: SDS Page, Infection, In Vitro

    6) Product Images from "Crystallographic Analysis of Polypyrimidine Tract-Binding Protein-Raver1 Interactions Involved in Regulation of Alternative Splicing"

    Article Title: Crystallographic Analysis of Polypyrimidine Tract-Binding Protein-Raver1 Interactions Involved in Regulation of Alternative Splicing

    Journal: Structure(London, England:1993)

    doi: 10.1016/j.str.2011.09.020

    Effect of Mutations of Raver1 PRIs on Binding to GST-PTB (A) Pull-down assays of binding of Raver1 PRI3 mutants to GST-PTB. Left: Loading controls for 35 S-Met-labeled PRI3-MS2 fusion proteins containing PRI3 wild-type (WT) and the mutants P6V, P6S, and P6A (see Experimental Procedures ). Right: Autoradiogram of PRI3-MS2 proteins pulled down with GST (1 μg) or GST-PTB (3 μg). (B) Pull-down assays of binding of Raver1 PRI4 mutants to GST-PTB. Left: Loading controls for 35 S-Met-labeled PRI4-MS2 fusion proteins. Right: GST pull-down of PRI4 constructs with GST (5 μg) or GST-PTB (15 μg). Although PRI4 constructs migrate as a doublet (as observed previously [ Rideau et al., 2006 ]), both products of the in vitro transcription-translation reaction contain the PRI since they bind PTB with the same affinity. (C) Comparison of binding of wild-type and mutant Raver1 PRIs to GST-PTB. Left: Loading controls. Right: GST pull-down of PRI4 constructs with GST (2 μg) or GST-PTB (6 μg). (D) PRI sequences from murine Raver1 (AAP33691), murine Raver2 (NP_898845), human matrin-3 (NP_001181884), and human hnRNP-L (NM_001533). Sequences shown for Raver1 are the 20 amino peptides used in pull-down assays; the shaded box indicates the sequences included in PRI-RRM2 chimeras for structural studies. Sequence similarity and identity within the PRI core are indicated. Residues in Raver1 PRI3 and PRI4 that were tested by mutagenesis are in boldface. See also Figure S3 .
    Figure Legend Snippet: Effect of Mutations of Raver1 PRIs on Binding to GST-PTB (A) Pull-down assays of binding of Raver1 PRI3 mutants to GST-PTB. Left: Loading controls for 35 S-Met-labeled PRI3-MS2 fusion proteins containing PRI3 wild-type (WT) and the mutants P6V, P6S, and P6A (see Experimental Procedures ). Right: Autoradiogram of PRI3-MS2 proteins pulled down with GST (1 μg) or GST-PTB (3 μg). (B) Pull-down assays of binding of Raver1 PRI4 mutants to GST-PTB. Left: Loading controls for 35 S-Met-labeled PRI4-MS2 fusion proteins. Right: GST pull-down of PRI4 constructs with GST (5 μg) or GST-PTB (15 μg). Although PRI4 constructs migrate as a doublet (as observed previously [ Rideau et al., 2006 ]), both products of the in vitro transcription-translation reaction contain the PRI since they bind PTB with the same affinity. (C) Comparison of binding of wild-type and mutant Raver1 PRIs to GST-PTB. Left: Loading controls. Right: GST pull-down of PRI4 constructs with GST (2 μg) or GST-PTB (6 μg). (D) PRI sequences from murine Raver1 (AAP33691), murine Raver2 (NP_898845), human matrin-3 (NP_001181884), and human hnRNP-L (NM_001533). Sequences shown for Raver1 are the 20 amino peptides used in pull-down assays; the shaded box indicates the sequences included in PRI-RRM2 chimeras for structural studies. Sequence similarity and identity within the PRI core are indicated. Residues in Raver1 PRI3 and PRI4 that were tested by mutagenesis are in boldface. See also Figure S3 .

    Techniques Used: Binding Assay, Labeling, Construct, In Vitro, Mutagenesis, Sequencing

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    Promega tnt coupled reticulate lysate system
    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. [ 62 ]). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. [ 63 ]). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnt t7 quick coupled transcription translation system
    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. [ 62 ]). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. [ 63 ]). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Tnt T7 Quick Coupled Transcription Translation System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega flag tagged onecut1 proteins
    <t>ONECUT1</t> shapes chromatin accessibility during PE-PP transition. (a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) ) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log 10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.
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    Promega transcription translation reaction protocol
    Polymerization and motility of WT and mutant actins and their interaction with CCT and actin-binding proteins. A , quantitation of in vitro <t>translation</t> <t>reaction</t> products shows significantly increased ratio of CCT-bound:released actin for R149C actin compared with WT actin (1.14 ± 0.47 versus 0.42 ± 0.1, p = 0.01) and R149C/N299T actin (0.64 ± 0.22, p = 0.048). Representative gel shown from six different assays. B , the actin concentration dependence of the polymerization rate of WT, N299T, and R149C/N299T monomers. See Table 2 for assembly and disassembly values. C , the polymerization rate of R149C/N299T in the absence or the presence of 3 μM profilin yielded a dissociation constant of the mutant actin for profilin of 2.7 μM, comparable to the value of 3.0 μM previously obtained for WT ( 7 ). The curve is the fit to the data as described previously ( 7 ). D , the actin polymerization rate of WT and R149C/N299T in the presence of 3 μM MRTF-A. The curves are fits to the data as described previously ( 8 ). Fit to the R149C/N299T data yields a K d of 3.3 μM and a Hill coefficient of 4.7, versus WT values of 1.8 μM and a Hill coefficient of 4.7. All WT data ( B – D ) are from previously published work ( 7 , 8 ). E , Gaussian distribution of speeds at which rhodamine phalloidin–stabilized actin (WT, N299T, or R149C/N299T) is moved by phosphorylated smooth muscle myosin in the absence ( upper panel ) or the presence ( lower panel ) of tropomyosin (Tpm 1.4). Speeds of large numbers of filaments were tracked with a semiautomated program and are provided in Table S3 . All pairs show statistically significant differences primarily because of the large dataset. Data were obtained using two protein preparations and two to four individual experiments. F , speed of movement using filaments that were not stabilized with phalloidin. Actin speeds were tracked manually and are provided in Table S3 . All pairs were statistically different except for WT versus N299T in the presence of Tpm1.4 ( p = 0.925). Data were obtained using two protein preparations and two individual experiments. Statistical significance was determined by one-way ANOVA followed by a Tukey's honest significant difference post hoc test. CCT, chaperonin-containing TCP-1; MRTF-A, myocardin-related <t>transcription</t> factor-A.
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    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. [ 62 ]). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. [ 63 ]). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Journal: Journal of Virology

    Article Title: Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

    doi: 10.1128/jvi.00488-22

    Figure Lengend Snippet: Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. [ 62 ]). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. [ 63 ]). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Article Snippet: Coupled in vitro transcription and translation reactions were carried out using the Promega TnT Coupled Reticulate Lysate System labeled with radioactive [35 S]methionine (PerkinElmer Inc.) according to the manufacturer’s protocol.

    Techniques: Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Sequencing, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Negative Control

    ONECUT1 shapes chromatin accessibility during PE-PP transition. (a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) ) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log 10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: ONECUT1 shapes chromatin accessibility during PE-PP transition. (a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) ) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log 10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Chromatin Immunoprecipitation, Activity Assay, Binding Assay

    Intrinsic defects in ONECUT1-depleted PP cells disturb launching of the β-cell program. (a,b) analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells ( a ) as well as PDX1 + /NKX6.1 + purified PP cells from HUES8 ONECUT1 KO and WT ( b . (c) ). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited ( ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test). {

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Intrinsic defects in ONECUT1-depleted PP cells disturb launching of the β-cell program. (a,b) analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells ( a ) as well as PDX1 + /NKX6.1 + purified PP cells from HUES8 ONECUT1 KO and WT ( b . (c) ). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited ( ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test). {"type":"entrez-geo","attrs":{"text":"GSE81547","term_id":"81547"}}

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Purification, RNA Sequencing Assay, Mass Spectrometry, Expressing, Real-time Polymerase Chain Reaction

    ONECUT1-depleted PSCs are defective in PP formation. (a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1 -p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images ( d ) and flow cytometry ( e ; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: ONECUT1-depleted PSCs are defective in PP formation. (a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1 -p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images ( d ) and flow cytometry ( e ; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Sequencing, Immunofluorescence, Western Blot, Flow Cytometry, Two Tailed Test

    Population ancestry studies of Family-1 and genetic analysis of ONECUT1 variants and T2D-associated SNPs. (a) . (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo , identified by WES in one patient. (g) T2D and BMI association in ONECUT1

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Population ancestry studies of Family-1 and genetic analysis of ONECUT1 variants and T2D-associated SNPs. (a) . (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo , identified by WES in one patient. (g) T2D and BMI association in ONECUT1

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Sequencing, Variant Assay, Targeted Sequencing

    Patient variant ONECUT1 -p.E231D impairs pancreatic differentiation in gene-edited HUES8 hESC. (a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1 -p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 -p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1 -p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Patient variant ONECUT1 -p.E231D impairs pancreatic differentiation in gene-edited HUES8 hESC. (a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1 -p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 -p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1 -p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Variant Assay, Generated, Sequencing, Immunofluorescence, Staining, Flow Cytometry, Two Tailed Test, Marker, Expressing, Immunoprecipitation, Western Blot, Over Expression

    DNA binding capacity of distinct ONECUT1 variants with clinical relevance. (a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: DNA binding capacity of distinct ONECUT1 variants with clinical relevance. (a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Binding Assay, Over Expression, Electro Mobility Shift Assay, Super-Shift Assay, FLAG-tag, In Vitro, Western Blot

    ONECUT1 protein-protein interaction requires its N-terminal end. (a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 ( b ) and NKX2.2 ( c ) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: ONECUT1 protein-protein interaction requires its N-terminal end. (a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 ( b ) and NKX2.2 ( c ) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Immunoprecipitation, Variant Assay, Western Blot, Over Expression

    ONECUT1-depleted PSCs are defective in PP formation. (a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) . Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and development al trajectories are indicated as borders and arrows, respectively. (f) of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) .

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: ONECUT1-depleted PSCs are defective in PP formation. (a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) . Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and development al trajectories are indicated as borders and arrows, respectively. (f) of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) .

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Derivative Assay, Immunofluorescence, Staining, Flow Cytometry, RNA Sequencing Assay, Expressing, Fluorescence, FACS, Purification

    Intrinsic defects in ONECUT1-depleted PP cells disturb the β-cell program. (a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 ( f ) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells ( g , n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Intrinsic defects in ONECUT1-depleted PP cells disturb the β-cell program. (a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 ( f ) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells ( g , n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Immunofluorescence, Staining, Flow Cytometry, Marker, Expressing

    Physical interaction between ONECUT1 and pancreatic transcription factors. (a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Physical interaction between ONECUT1 and pancreatic transcription factors. (a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Luciferase, Reporter Assay, Construct, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Genome Wide, Immunoprecipitation, Over Expression

    Cooperative ONECUT1 interaction at putative enhancers. (a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2 , and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in ( c ). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Cooperative ONECUT1 interaction at putative enhancers. (a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2 , and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in ( c ). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, Chromatin Immunoprecipitation, Luciferase, Reporter Assay

    Fine-mapped T2D-associated variants reside at ONECUT1 locus. (a) and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value

    Journal: Nature medicine

    Article Title: ONECUT1 mutations and variants in diabetes

    doi: 10.1038/s41591-021-01502-7

    Figure Lengend Snippet: Fine-mapped T2D-associated variants reside at ONECUT1 locus. (a) and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value

    Article Snippet: In vitro translated Flag-tagged ONECUT1 proteins (TnT® Quick Coupled Transcription/Translation System, Promega) were used for electromobility gel shift assays in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol.

    Techniques: Chromatin Immunoprecipitation

    Polymerization and motility of WT and mutant actins and their interaction with CCT and actin-binding proteins. A , quantitation of in vitro translation reaction products shows significantly increased ratio of CCT-bound:released actin for R149C actin compared with WT actin (1.14 ± 0.47 versus 0.42 ± 0.1, p = 0.01) and R149C/N299T actin (0.64 ± 0.22, p = 0.048). Representative gel shown from six different assays. B , the actin concentration dependence of the polymerization rate of WT, N299T, and R149C/N299T monomers. See Table 2 for assembly and disassembly values. C , the polymerization rate of R149C/N299T in the absence or the presence of 3 μM profilin yielded a dissociation constant of the mutant actin for profilin of 2.7 μM, comparable to the value of 3.0 μM previously obtained for WT ( 7 ). The curve is the fit to the data as described previously ( 7 ). D , the actin polymerization rate of WT and R149C/N299T in the presence of 3 μM MRTF-A. The curves are fits to the data as described previously ( 8 ). Fit to the R149C/N299T data yields a K d of 3.3 μM and a Hill coefficient of 4.7, versus WT values of 1.8 μM and a Hill coefficient of 4.7. All WT data ( B – D ) are from previously published work ( 7 , 8 ). E , Gaussian distribution of speeds at which rhodamine phalloidin–stabilized actin (WT, N299T, or R149C/N299T) is moved by phosphorylated smooth muscle myosin in the absence ( upper panel ) or the presence ( lower panel ) of tropomyosin (Tpm 1.4). Speeds of large numbers of filaments were tracked with a semiautomated program and are provided in Table S3 . All pairs show statistically significant differences primarily because of the large dataset. Data were obtained using two protein preparations and two to four individual experiments. F , speed of movement using filaments that were not stabilized with phalloidin. Actin speeds were tracked manually and are provided in Table S3 . All pairs were statistically different except for WT versus N299T in the presence of Tpm1.4 ( p = 0.925). Data were obtained using two protein preparations and two individual experiments. Statistical significance was determined by one-way ANOVA followed by a Tukey's honest significant difference post hoc test. CCT, chaperonin-containing TCP-1; MRTF-A, myocardin-related transcription factor-A.

    Journal: The Journal of Biological Chemistry

    Article Title: Resistance of Acta2R149C/+ mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex

    doi: 10.1016/j.jbc.2021.101228

    Figure Lengend Snippet: Polymerization and motility of WT and mutant actins and their interaction with CCT and actin-binding proteins. A , quantitation of in vitro translation reaction products shows significantly increased ratio of CCT-bound:released actin for R149C actin compared with WT actin (1.14 ± 0.47 versus 0.42 ± 0.1, p = 0.01) and R149C/N299T actin (0.64 ± 0.22, p = 0.048). Representative gel shown from six different assays. B , the actin concentration dependence of the polymerization rate of WT, N299T, and R149C/N299T monomers. See Table 2 for assembly and disassembly values. C , the polymerization rate of R149C/N299T in the absence or the presence of 3 μM profilin yielded a dissociation constant of the mutant actin for profilin of 2.7 μM, comparable to the value of 3.0 μM previously obtained for WT ( 7 ). The curve is the fit to the data as described previously ( 7 ). D , the actin polymerization rate of WT and R149C/N299T in the presence of 3 μM MRTF-A. The curves are fits to the data as described previously ( 8 ). Fit to the R149C/N299T data yields a K d of 3.3 μM and a Hill coefficient of 4.7, versus WT values of 1.8 μM and a Hill coefficient of 4.7. All WT data ( B – D ) are from previously published work ( 7 , 8 ). E , Gaussian distribution of speeds at which rhodamine phalloidin–stabilized actin (WT, N299T, or R149C/N299T) is moved by phosphorylated smooth muscle myosin in the absence ( upper panel ) or the presence ( lower panel ) of tropomyosin (Tpm 1.4). Speeds of large numbers of filaments were tracked with a semiautomated program and are provided in Table S3 . All pairs show statistically significant differences primarily because of the large dataset. Data were obtained using two protein preparations and two to four individual experiments. F , speed of movement using filaments that were not stabilized with phalloidin. Actin speeds were tracked manually and are provided in Table S3 . All pairs were statistically different except for WT versus N299T in the presence of Tpm1.4 ( p = 0.925). Data were obtained using two protein preparations and two individual experiments. Statistical significance was determined by one-way ANOVA followed by a Tukey's honest significant difference post hoc test. CCT, chaperonin-containing TCP-1; MRTF-A, myocardin-related transcription factor-A.

    Article Snippet: The individual SM α-actin variants were expressed in vitro in reticulocyte lysates using the Promega Transcription Translation Reaction Protocol (400 ng DNA) and labeled with 35S methionine.

    Techniques: Mutagenesis, Binding Assay, Quantitation Assay, In Vitro, Concentration Assay