helascribe® nuclear extract in vitro transcription grade  (Promega)

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.

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: 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.

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: 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).

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: 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).

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: 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).

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: 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).

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: 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.

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: 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).

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

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