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  • 99
    New England Biolabs t4 polynucleotide kinase
    Retroposon- and repeat-derived siRNAs have modified 5′ and 3′ termini. ( A ) A synthetic 30-nt RNA (lane 1) was sequentially treated with <t>T4</t> polynucleotide kinase (PNK, lane 2) and calf intestinal alkaline phosphatase (CIP, lane 3), separated
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 pnk
    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by <t>T4</t> PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
    T4 Pnk, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 814 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bangalore Genei t4 polynucleotide kinase
    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by <t>T4</t> PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
    T4 Polynucleotide Kinase, supplied by Bangalore Genei, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc t4 polynucleotide kinase
    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by <t>T4</t> PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
    T4 Polynucleotide Kinase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega t4 polynucleotide kinase
    Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using <t>T4</t> polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.
    T4 Polynucleotide Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 5420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa t4 pnk
    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with <t>T4</t> PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    T4 Pnk, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Toyobo t4 polynucleotide kinase
    ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and <t>T4</t> polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).
    T4 Polynucleotide Kinase, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 748 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche t4 polynucleotide kinase
    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by <t>T4</t> polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).
    T4 Polynucleotide Kinase, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzymatics t4 polynucleotide kinase
    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by <t>T4</t> polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).
    T4 Polynucleotide Kinase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim t4 polynucleotide kinase
    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with <t>T4</t> polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).
    T4 Polynucleotide Kinase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 532 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa 10x t4 pnk buffer
    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with <t>T4</t> polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).
    10x T4 Pnk Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare t4 polynucleotide kinase
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    T4 Polynucleotide Kinase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 2055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer t4 polynucleotide kinase
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    T4 Polynucleotide Kinase, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore t4 pnk
    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
    T4 Pnk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa t4 pnk enzyme
    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
    T4 Pnk Enzyme, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzymatics t4 pnk buffer
    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
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    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
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    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
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    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
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    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
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    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
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    Image Search Results


    Retroposon- and repeat-derived siRNAs have modified 5′ and 3′ termini. ( A ) A synthetic 30-nt RNA (lane 1) was sequentially treated with T4 polynucleotide kinase (PNK, lane 2) and calf intestinal alkaline phosphatase (CIP, lane 3), separated

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei

    doi: 10.1073/pnas.0907766106

    Figure Lengend Snippet: Retroposon- and repeat-derived siRNAs have modified 5′ and 3′ termini. ( A ) A synthetic 30-nt RNA (lane 1) was sequentially treated with T4 polynucleotide kinase (PNK, lane 2) and calf intestinal alkaline phosphatase (CIP, lane 3), separated

    Article Snippet: Small RNAs were size-selected ( ) and treated with a variety of enzymes under manufacturer-recommended conditions: Calf intestinal alkaline phosphatase (CIP; Amersham) and T4 polynucleotide kinase (T4 PNK; New England Biolabs) assays were carried out for 1 h at 37 °C, and Terminator exonuclease (Epicentre) was added for 1 h at 30 °C.

    Techniques: Derivative Assay, Modification

    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

    doi: 10.1016/j.omtn.2017.07.008

    Figure Lengend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Article Snippet: The protocol was modified as follows when CIP or T4 PNK treatment is necessary: (1) for CIP treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of CIP, 4 μL of 10× CutSmart buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min; and (2) for T4 PNK treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of T4 PNK, 4 μL of 10× T4 PNK buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min. All T7 in vitro transcription products were purified by Micro Bio-Spin P-30 Gel Columns, Tris Buffer, from Bio-Rad.

    Techniques: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells

    doi: 10.1681/ASN.2018090914

    Figure Lengend Snippet: RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Article Snippet: Beads were resuspended in PNK buffer containing 5 mM DTT, 0.2 μ Ci/ μ l ( γ 32 P)ATP (Hartmann-Analytic), and 1 U/ μ l T4 PNK (ThermoFisher).

    Techniques: Selection, Software, RNA Binding Assay, Expressing, Imaging, Clone Assay, Immunoprecipitation, Labeling, Autoradiography, Western Blot

    Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

    Journal: Journal of Bacteriology

    Article Title: Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †

    doi: 10.1128/JB.00011-07

    Figure Lengend Snippet: Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

    Article Snippet: The purified DNA fragments (3.5 pmol) were end labeled with 10 U of T4 polynucleotide kinase (Promega) and 10 μCi of [γ32 -P]ATP (Perkin-Elmer, Wellesley, MA) at 37°C for 10 min.

    Techniques: In Vivo, Binding Assay, Sequencing, Labeling, End Labeling, Polyacrylamide Gel Electrophoresis

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Article Snippet: T4 PNK consists of four identical subunits of 28.9 kDa each.

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Article Snippet: T4 PNK consists of four identical subunits of 28.9 kDa each.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and T4 polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).

    Journal: Nucleic Acids Research

    Article Title: Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm

    doi: 10.1093/nar/gki309

    Figure Lengend Snippet: ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and T4 polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).

    Article Snippet: Preparation of RNA fragments, 5′ end phosphorylation using T4 polynucleotide kinase (Toyobo), 3′ end-nucleoside deprivation using NaIO4 , and dephosphorylation using E.coli alkaline phosphatase (BAP) (Takara Shuzo) were performed as described ( ).

    Techniques: Thin Layer Chromatography, Modification, Generated, Labeling

    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Journal: RNA

    Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

    doi: 10.1261/rna.064501.117

    Figure Lengend Snippet: cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Article Snippet: For 5′-end labeling, degraded RNA fragments, γ-P32 -ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32 ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min.

    Techniques: Labeling, Marker

    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with T4 polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).

    Journal: Nucleic Acids Research

    Article Title: RecJ exonuclease: substrates, products and interaction with SSB

    doi: 10.1093/nar/gkj503

    Figure Lengend Snippet: RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with T4 polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).

    Article Snippet: Oligonucleotide A was 5′ labeled using T4 polynucleotide kinase and [γ-33 P]ATP and purified by chromatography with G-50 Sephadex (Boehringer Mannheim), phenol/chloroform extraction, ethanol precipitation and resuspended in TE buffer (pH 8.0) or 10 mM Tris–HCl (pH 8.0).

    Techniques: Labeling, Purification

    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by T4 polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.

    Journal: American journal of physiology. Gastrointestinal and liver physiology

    Article Title: Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1?

    doi: 10.1152/ajpgi.00121.2004

    Figure Lengend Snippet: EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by T4 polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.

    Article Snippet: Briefly, 3.5 pmol of the probe was incubated with 1 µl of T4 polynucleotide kinase 10× buffer (700 mM Tris·HCl, pH 7.6, 100 mM MgCl2 , 50 mM DTT), 1 µl of [γ-32 P]ATP (3,000 Ci/mmol at 10 mCi/ml, Amersham Biosciences, Piscataway, NJ), and 1 µl of T4 polynucleotide kinase (5–10 U/µl) in a 10 µl reaction at 37°C for 10 min.

    Techniques: Binding Assay, Labeling, Electrophoretic Mobility Shift Assay, Incubation

    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Journal: Nature Communications

    Article Title: Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    doi: 10.1038/ncomms9430

    Figure Lengend Snippet: Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Article Snippet: To measure the half-life of saiRNA with terminal 2′, 3′-cyclic phosphate or hydroxyl groups, T7-transcribed saiRNA-RZs treated or untreated with T4 PNK were incubated with Ago2-KO 293 cell lysates in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 1% NP-40, 0.5% sodium deoxycholate (Sigma), 0.1 U μl−1 RNase Inhibitor and 1/100 protease inhibitor cocktail) for different time periods.

    Techniques: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, shRNA, Luciferase, Northern Blot, Western Blot, Transfection, Activity Assay, Stable Transfection, Transduction