Fig. 4.
Fig. 4. Experiments with microcircles. (A) Nuclease digestion experiments. Radiolabeled linear 27-bp HRE linear probes or microcircles containing HRE sequences were incubated with S1 nuclease (1 U, 15 minutes, 37°C) or Bal 31 nuclease (1 U, 15 minutes, 37°C), then analyzed by PAGE and autoradiography. Lane 1, untreated linear probe. Lane 2, untreated microcircles after ligation reaction and before gel purification. The arrow indicates unincorporated residual linear probe. Lanes 3 and 4, linear probe after digestion with S1 nuclease or Bal 31 nuclease, respectively. Lanes 5 and 6, microcircles after digestion with S1 nuclease or Bal 31 nuclease, respectively. (B) Mobility shift assays with nuclear extracts. Radiolabeled HRE microcircles were incubated with nuclear extracts from hemin-treated U937 cells (12 μg) or with bovine serum albumin, 4 to 80 μg, then analyzed by agarose gel electrophoresis and autoradiography. Lane 1, free microcircle probe. Lanes 2 and 3, probe plus nuclear extracts. Lanes 4, 5, 6, and 7, probe plus BSA, 4, 8, 40, or 80 μg, respectively. (C) Cold competition experiments. Gel mobility shift assays were performed with radiolabeled HRE microcircle probes. Competition experiments include a 15-minute preincubation with increasing concentrations of unlabeled linear oligonucleotides, either authentic 27-bp HRE, or scrambled HRE sequences. Panel I: Competition study with authentic probe. Lane 1, free microcircle probe. Lanes 2 and 3, probe plus nuclear extracts. Lanes 4, 5, and 6, probe, nuclear extract plus 50-fold, 100-fold, or 250-fold excess of linear 27-bp HRE. Panel II: Competition study with irrelevant probe. Lane 1, free microcircle probe; Lane 2, probe plus nuclear extract. Lanes 3, 4, and 5, probe, nuclear extract, plus 50-fold, 100-fold, or 200-fold excess of linear scrambled HRE. (D) Immunoblot analyses of gel shift bonds. Radiolabeled HRE microcircles were incubated with 12, 40, or 80 μg of nuclear extract from hemin-treated U937 cells, then analyzed by agarose gel electrophoresis. Panel I: Analysis by autoradiography (48-hour exposure). Lane 1, free microcircle probe. Lanes 2, 3, and 4, probe plus 12, 40, or 80 μg of nuclear extract. Panel II: Immunoblot analysis with anti-KuAg (Ab-3) (chemiluminescence exposure, 2 minutes). Lanes 1 to 4, same as panel I. Panel III: Immunoblot analysis with mouse anti–c-myc IgG (chemiluminescence exposure, 2 minutes). Lanes 1 to 4, same as panels I and II.

Experiments with microcircles. (A) Nuclease digestion experiments. Radiolabeled linear 27-bp HRE linear probes or microcircles containing HRE sequences were incubated with S1 nuclease (1 U, 15 minutes, 37°C) or Bal 31 nuclease (1 U, 15 minutes, 37°C), then analyzed by PAGE and autoradiography. Lane 1, untreated linear probe. Lane 2, untreated microcircles after ligation reaction and before gel purification. The arrow indicates unincorporated residual linear probe. Lanes 3 and 4, linear probe after digestion with S1 nuclease or Bal 31 nuclease, respectively. Lanes 5 and 6, microcircles after digestion with S1 nuclease or Bal 31 nuclease, respectively. (B) Mobility shift assays with nuclear extracts. Radiolabeled HRE microcircles were incubated with nuclear extracts from hemin-treated U937 cells (12 μg) or with bovine serum albumin, 4 to 80 μg, then analyzed by agarose gel electrophoresis and autoradiography. Lane 1, free microcircle probe. Lanes 2 and 3, probe plus nuclear extracts. Lanes 4, 5, 6, and 7, probe plus BSA, 4, 8, 40, or 80 μg, respectively. (C) Cold competition experiments. Gel mobility shift assays were performed with radiolabeled HRE microcircle probes. Competition experiments include a 15-minute preincubation with increasing concentrations of unlabeled linear oligonucleotides, either authentic 27-bp HRE, or scrambled HRE sequences. Panel I: Competition study with authentic probe. Lane 1, free microcircle probe. Lanes 2 and 3, probe plus nuclear extracts. Lanes 4, 5, and 6, probe, nuclear extract plus 50-fold, 100-fold, or 250-fold excess of linear 27-bp HRE. Panel II: Competition study with irrelevant probe. Lane 1, free microcircle probe; Lane 2, probe plus nuclear extract. Lanes 3, 4, and 5, probe, nuclear extract, plus 50-fold, 100-fold, or 200-fold excess of linear scrambled HRE. (D) Immunoblot analyses of gel shift bonds. Radiolabeled HRE microcircles were incubated with 12, 40, or 80 μg of nuclear extract from hemin-treated U937 cells, then analyzed by agarose gel electrophoresis. Panel I: Analysis by autoradiography (48-hour exposure). Lane 1, free microcircle probe. Lanes 2, 3, and 4, probe plus 12, 40, or 80 μg of nuclear extract. Panel II: Immunoblot analysis with anti-KuAg (Ab-3) (chemiluminescence exposure, 2 minutes). Lanes 1 to 4, same as panel I. Panel III: Immunoblot analysis with mouse anti–c-myc IgG (chemiluminescence exposure, 2 minutes). Lanes 1 to 4, same as panels I and II.

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