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Promega hela cell nuclear extracts in vitro transcription grade

<t>HeLa</t> cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Hela Cell Nuclear Extracts In Vitro Transcription Grade, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hela cell nuclear extracts in vitro transcription grade - by Bioz Stars, 2026-09

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1) Product Images from "Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences * "

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

Journal: The Journal of biological chemistry

doi:

HeLa cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Figure Legend Snippet: HeLa cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Techniques Used: Confocal Microscopy

Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °C with either the Fl-PNA conjugated 4.2-kb plasmid pUSAG3 (A–D) or the Fl-PNA conjugated 14.4-kb plasmid pUSAG9 (E–H), both at 10 µg/ml, in the presence or absence of cellular extracts. BSA was added to 10 mg/ml to cells incubated in the absence of extracts (A and E). HeLa cell nuclear extract (Promega) was added to 0.5 mg/ml B and F). HeLa cell cytoplasmic extract was added to 5 mg/ml in the absence (C and G) or presence (D and H) of nuclear extract.

Figure Legend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °C with either the Fl-PNA conjugated 4.2-kb plasmid pUSAG3 (A–D) or the Fl-PNA conjugated 14.4-kb plasmid pUSAG9 (E–H), both at 10 µg/ml, in the presence or absence of cellular extracts. BSA was added to 10 mg/ml to cells incubated in the absence of extracts (A and E). HeLa cell nuclear extract (Promega) was added to 0.5 mg/ml B and F). HeLa cell cytoplasmic extract was added to 5 mg/ml in the absence (C and G) or presence (D and H) of nuclear extract.

Techniques Used: Incubation, Plasmid Preparation

Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °Cwith transport buffer lacking ATP, phosphocreatine, creatine phosphokinase, GTP, and Fl-PNA/pUSAG3 (10 µg/ml). The cells were then washed, fixed with 3% paraformaldehyde, and viewed by confocal microscopy. Cells incubated in the absence of cellular extracts contained 10 mg/ml BSA (A). Nuclear (B and D) and cytoplasmic (C and D) extracts were depleted of endogenous nucleotide triphosphates by preincubating the extracts with 10 units/ml apyrase at 25 °C for 30 min.

Figure Legend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °Cwith transport buffer lacking ATP, phosphocreatine, creatine phosphokinase, GTP, and Fl-PNA/pUSAG3 (10 µg/ml). The cells were then washed, fixed with 3% paraformaldehyde, and viewed by confocal microscopy. Cells incubated in the absence of cellular extracts contained 10 mg/ml BSA (A). Nuclear (B and D) and cytoplasmic (C and D) extracts were depleted of endogenous nucleotide triphosphates by preincubating the extracts with 10 units/ml apyrase at 25 °C for 30 min.

Techniques Used: Incubation, Confocal Microscopy

Permeabilized HeLa cells were incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml) for 4 h at 37 °C. All reactions contained HeLa cytoplasm at 5 mg/ml and either no additional lectin (A), concanavalin A (0.1 mg/ml; B) or wheat germ agglutinin (0.1 mg/ml; C).

Figure Legend Snippet: Permeabilized HeLa cells were incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml) for 4 h at 37 °C. All reactions contained HeLa cytoplasm at 5 mg/ml and either no additional lectin (A), concanavalin A (0.1 mg/ml; B) or wheat germ agglutinin (0.1 mg/ml; C).

Techniques Used: Incubation

HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSA-NLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. His-tagged importin α, importin β, and RAN were purified by nickel affinity chromatography and concentrated for addition to the reactions. The reactions contained BSA alone (10 mg/ml; A and E), nuclear extract alone (0.5 mg/ml; B and F), importin α, importin β, and RAN (0.5 mg/ml each; C and G), or importin α, importin β, RAN, and nuclear extract (D and H).

Figure Legend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSA-NLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. His-tagged importin α, importin β, and RAN were purified by nickel affinity chromatography and concentrated for addition to the reactions. The reactions contained BSA alone (10 mg/ml; A and E), nuclear extract alone (0.5 mg/ml; B and F), importin α, importin β, and RAN (0.5 mg/ml each; C and G), or importin α, importin β, RAN, and nuclear extract (D and H).

Techniques Used: Incubation, Purification, Affinity Chromatography

Figure Legend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSANLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. All reactions contained HeLa cell nuclear extract (0.5 mg/ml). Affinity purified His-tagged importins and RAN were added to 0.5 mg/ml each. The reactions contained importin α, importin β, and RAN (A and E), importin α, and RAN (B and F), importin β, and RAN (C and G) or importin α and importin β (D and H).

Techniques Used: Incubation, Affinity Purification

Permeabilized HeLa cells were incubated for 4 h at 37 °C with transport buffer containing HeLa cytoplasmic extract (5 mg/ml) and Fl-PNA/pUSAG3 (10 µg/ml) in the absence (A) or presence of a 1000-fold molar excess of BSA-NLS (0.23 mg/ml; B), a 100-fold excess of pUC19 (0.7 mg/ml; C), or a 100-fold molar excess of SV40 DNA (1.3 mg/ml; D).

Figure Legend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C with transport buffer containing HeLa cytoplasmic extract (5 mg/ml) and Fl-PNA/pUSAG3 (10 µg/ml) in the absence (A) or presence of a 1000-fold molar excess of BSA-NLS (0.23 mg/ml; B), a 100-fold excess of pUC19 (0.7 mg/ml; C), or a 100-fold molar excess of SV40 DNA (1.3 mg/ml; D).

Techniques Used: Incubation

Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3DSV40 (C and D), both present at 10 µg/ml.

Figure Legend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3DSV40 (C and D), both present at 10 µg/ml.

Techniques Used: Incubation

Plasmid DNA (0.1 µg) was incubated for 4 h at 37 °C with HeLa cell cytoplasmic extract (5 mg/ml) in transport buffer. Protein was removed by phenol: chloroform extraction, and the DNAs were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed using a mixture of 32P-labeled pUSAG3 and pGenegrip blank. Lanes 1–3 contain 0.1 µg of unincubated plasmid used as starting material, and lanes 4–6 contain DNA incubated with cytoplasmic extract. Three plasmids were used: Fl-PNA/pUSAG3 (lanes 1 and 4), rhodamine-labeled PNA/ pGenegrip-GFP (lanes 2 and 5), and unlabeled pUSAG3 (lanes 3 and 6).

Figure Legend Snippet: Plasmid DNA (0.1 µg) was incubated for 4 h at 37 °C with HeLa cell cytoplasmic extract (5 mg/ml) in transport buffer. Protein was removed by phenol: chloroform extraction, and the DNAs were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed using a mixture of 32P-labeled pUSAG3 and pGenegrip blank. Lanes 1–3 contain 0.1 µg of unincubated plasmid used as starting material, and lanes 4–6 contain DNA incubated with cytoplasmic extract. Three plasmids were used: Fl-PNA/pUSAG3 (lanes 1 and 4), rhodamine-labeled PNA/ pGenegrip-GFP (lanes 2 and 5), and unlabeled pUSAG3 (lanes 3 and 6).

Techniques Used: Plasmid Preparation, Incubation, Extraction, Agarose Gel Electrophoresis, Membrane, Labeling

In Vitro:

Article Title: The role of Sp1 and Sp3 in the constitutive DPYD gene expression.
Article Snippet: Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in the 5-fluorouracil (5-FU) catabolic pathway, has been implicated as one of the factors determining the efficacy and toxicity of the anticancer agent 5-FU.. Studies have attributed variation in DPD activity partially to alterations at the transcriptional level of DPYD gene.. We investigated the transcription factors implicated in the constitutive expression of DPYD by utilizing a 174-bp fragment of the DPYD promoter region in which three consensus Sp protein binding sites (SpA, SpB and SpC) were predicted.

Article Title: Patching and Single-Strand Ligation in Nonhomologous DNA End Joining Despite Persistence of a Closely Opposed 3?-Phosphoglycolate-Terminated Strand Break
Article Snippet: .. Complete repair of both strands (DS) by PG removal, end alignment/annealing, patching ... End-Joining Reactions Reactions contained HeLa cell nuclear extract (27 μg protein, Promega in vitro transcription grade), 0.1 μg of X4L4 (Trevigen), 50 m M triethanolammonium acetate, 10 m M Tris HCl, pH 7.9, 1 m M Mg(OAc) 2 , 40 m M KOAc, 0.5 m M dithiothreitol, 1 m M ATP, 50 μ M of each dNTP or dideoxynucleoside 5′-triphosphate (ddNTP), 50 μg/ml BSA, and 20 ng DNA substrate in a total volume of 16 μl. ..

Article Title: Patching and Single-Strand Ligation in Nonhomologous DNA End Joining Despite Persistence of a Closely Opposed 3?-Phosphoglycolate-Terminated Strand Break
Article Snippet: .. Reactions contained HeLa cell nuclear extract (27 μg protein, Promega in vitro transcription grade), 0.1 μg of X4L4 (Trevigen), 50 m M triethanolammonium acetate, 10 m M Tris HCl, pH 7.9, 1 m M Mg(OAc) 2 , 40 m M KOAc, 0.5 m M dithiothreitol, 1 m M ATP, 50 μ M of each dNTP or dideoxynucleoside 5′-triphosphate (ddNTP), 50 μg/ml BSA, and 20 ng DNA substrate in a total volume of 16 μl. ..

Article Title: Patching and Single-Strand Ligation in Nonhomologous DNA End Joining Despite Persistence of a Closely Opposed 3′-Phosphoglycolate-Terminated Strand Break
Article Snippet: All other enzymes were from New England Biolabs and were used in the buffers provided. .. Reactions contained HeLa cell nuclear extract (27 mg protein, Promega in vitro transcription grade), 0.1 mg of X4L4 (Trevigen), 50 mM triethanolammonium acetate, 10 mM Tris HCl, pH 7.9, 1 mM Mg(OAc)2, 40 mM KOAc, 0.5 mM dithiothreitol, 1 mM ATP, 50 mM of each dNTP or dideoxynucleoside 59-triphosphate (ddNTP), 50 mg/ml BSA, and 20 ng DNA substrate in a total volume of 16 ml. ..

Article Title: Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase λ in human nuclear extracts
Article Snippet: .. Reaction mixtures contained HeLa nuclear extract (27 μg protein, in vitro transcription grade, Promega, Madison, WI), 0.1 μg of XRCC4/LigIV (Trevigen, Gaithersburg, MD), 50 mM triethanolamine–NaOH, 10 mM Tris–HCl pH 7.9, 1 mM Mg(OAc) 2 , 40 mM KOAc, 0.5 mM dithiothreitol, 1 mM ATP, 50 μM of each dNTP or dideoxynucleoside 5′-triphosphate (ddNTP), 50 μg/ml BSA and 16 ng DNA substrate in a total volume of 16 μl. ..

Image Search Results

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: HeLa cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Article Snippet: Where indicated, cytoplasmic extracts prepared from HeLa cells ( 7 ) were added to between 5 and 7 mg/ml, HeLa cell nuclear extracts ( in vitro transcription grade, Promega, Madison, WI) were added to 0.25 mg/ml, and affinity purified, recombinant his-tagged RAN, importin α, and importin β were used at 0.5 mg/ml each.

Techniques: Confocal Microscopy

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °C with either the Fl-PNA conjugated 4.2-kb plasmid pUSAG3 (A–D) or the Fl-PNA conjugated 14.4-kb plasmid pUSAG9 (E–H), both at 10 µg/ml, in the presence or absence of cellular extracts. BSA was added to 10 mg/ml to cells incubated in the absence of extracts (A and E). HeLa cell nuclear extract (Promega) was added to 0.5 mg/ml B and F). HeLa cell cytoplasmic extract was added to 5 mg/ml in the absence (C and G) or presence (D and H) of nuclear extract.

Techniques: Incubation, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °Cwith transport buffer lacking ATP, phosphocreatine, creatine phosphokinase, GTP, and Fl-PNA/pUSAG3 (10 µg/ml). The cells were then washed, fixed with 3% paraformaldehyde, and viewed by confocal microscopy. Cells incubated in the absence of cellular extracts contained 10 mg/ml BSA (A). Nuclear (B and D) and cytoplasmic (C and D) extracts were depleted of endogenous nucleotide triphosphates by preincubating the extracts with 10 units/ml apyrase at 25 °C for 30 min.

Techniques: Incubation, Confocal Microscopy

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Permeabilized HeLa cells were incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml) for 4 h at 37 °C. All reactions contained HeLa cytoplasm at 5 mg/ml and either no additional lectin (A), concanavalin A (0.1 mg/ml; B) or wheat germ agglutinin (0.1 mg/ml; C).

Techniques: Incubation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSA-NLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. His-tagged importin α, importin β, and RAN were purified by nickel affinity chromatography and concentrated for addition to the reactions. The reactions contained BSA alone (10 mg/ml; A and E), nuclear extract alone (0.5 mg/ml; B and F), importin α, importin β, and RAN (0.5 mg/ml each; C and G), or importin α, importin β, RAN, and nuclear extract (D and H).

Techniques: Incubation, Purification, Affinity Chromatography

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSANLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. All reactions contained HeLa cell nuclear extract (0.5 mg/ml). Affinity purified His-tagged importins and RAN were added to 0.5 mg/ml each. The reactions contained importin α, importin β, and RAN (A and E), importin α, and RAN (B and F), importin β, and RAN (C and G) or importin α and importin β (D and H).

Techniques: Incubation, Affinity Purification

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C with transport buffer containing HeLa cytoplasmic extract (5 mg/ml) and Fl-PNA/pUSAG3 (10 µg/ml) in the absence (A) or presence of a 1000-fold molar excess of BSA-NLS (0.23 mg/ml; B), a 100-fold excess of pUC19 (0.7 mg/ml; C), or a 100-fold molar excess of SV40 DNA (1.3 mg/ml; D).

Techniques: Incubation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3DSV40 (C and D), both present at 10 µg/ml.

Techniques: Incubation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Plasmid DNA (0.1 µg) was incubated for 4 h at 37 °C with HeLa cell cytoplasmic extract (5 mg/ml) in transport buffer. Protein was removed by phenol: chloroform extraction, and the DNAs were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed using a mixture of 32P-labeled pUSAG3 and pGenegrip blank. Lanes 1–3 contain 0.1 µg of unincubated plasmid used as starting material, and lanes 4–6 contain DNA incubated with cytoplasmic extract. Three plasmids were used: Fl-PNA/pUSAG3 (lanes 1 and 4), rhodamine-labeled PNA/ pGenegrip-GFP (lanes 2 and 5), and unlabeled pUSAG3 (lanes 3 and 6).

Techniques: Plasmid Preparation, Incubation, Extraction, Agarose Gel Electrophoresis, Membrane, Labeling

End joining of a substrate containing a thymine glycol base. ( A ) Schematic of the internally labeled (asterisk) substrate, showing proposed mechanisms of formation of products giving 43-, 42- and 35-base BstXI/AvaI fragments. The italicized T is the site of thymine glycol substitution. Bolded nucleotides indicate gap filling. The 35-base product is shown as arising by 3′→5′ resection, but it could in principle be generated by 5′→3′ resection as well. ( B ) Unmodified or thymine glycol-substituted substrate was incubated for 6 h with X4L4-supplemented HeLa nuclear extracts that had been immunodepleted of polλ or mock depleted. Polλ or polμ (70 ng) was added as indicated. ( C ) Same as (B), except extracts were not immunodepleted, polλ was added to all samples and samples contained either ddCTP or ddTTP in place of normal dNTPs as indicated. ( D ) Quantitative data from (B) and similar experiments; the abundance of each product is normalized to the total of all head-to-tail end-joining products in the sample with the unmodified substrate (‘T’) and added polλ; ‘T-glycol’ = thymine glycol-substituted substrate. Error bars show mean and SE of 3–4 experiments. ( E ) Similar quantitation of data from (C) and a replicate experiment. Error bars show the range of values obtained in the two experiments.

Journal: Nucleic Acids Research

Article Title: Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase λ in human nuclear extracts

doi: 10.1093/nar/gkn126

Figure Lengend Snippet: End joining of a substrate containing a thymine glycol base. ( A ) Schematic of the internally labeled (asterisk) substrate, showing proposed mechanisms of formation of products giving 43-, 42- and 35-base BstXI/AvaI fragments. The italicized T is the site of thymine glycol substitution. Bolded nucleotides indicate gap filling. The 35-base product is shown as arising by 3′→5′ resection, but it could in principle be generated by 5′→3′ resection as well. ( B ) Unmodified or thymine glycol-substituted substrate was incubated for 6 h with X4L4-supplemented HeLa nuclear extracts that had been immunodepleted of polλ or mock depleted. Polλ or polμ (70 ng) was added as indicated. ( C ) Same as (B), except extracts were not immunodepleted, polλ was added to all samples and samples contained either ddCTP or ddTTP in place of normal dNTPs as indicated. ( D ) Quantitative data from (B) and similar experiments; the abundance of each product is normalized to the total of all head-to-tail end-joining products in the sample with the unmodified substrate (‘T’) and added polλ; ‘T-glycol’ = thymine glycol-substituted substrate. Error bars show mean and SE of 3–4 experiments. ( E ) Similar quantitation of data from (C) and a replicate experiment. Error bars show the range of values obtained in the two experiments.

Article Snippet: Reaction mixtures contained HeLa nuclear extract (27 μg protein, in vitro transcription grade, Promega, Madison, WI), 0.1 μg of XRCC4/LigIV (Trevigen, Gaithersburg, MD), 50 mM triethanolamine–NaOH, 10 mM Tris–HCl pH 7.9, 1 mM Mg(OAc) 2 , 40 mM KOAc, 0.5 mM dithiothreitol, 1 mM ATP, 50 μM of each dNTP or dideoxynucleoside 5′-triphosphate (ddNTP), 50 μg/ml BSA and 16 ng DNA substrate in a total volume of 16 μl.

Techniques: Labeling, Generated, Incubation, Quantitation Assay

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hela cell nuclear extracts in vitro transcription grade (Promega)

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