Figure 5.
Figure 5. Transfection of Ku80 to HeLa cells. (A) Expression of Ku80 on HeLa cells transfected with pKu80. Ku80 cDNA was inserted to expression plasmid pcD and the resulted pKu80 was transfected to HeLa cells (HeLa-Ku80) using lipofectin. Empty pcD was used for a mock transfection (HeLa-mock). The transfected cells (2 × 105) were incubated with 5 μg/mL of each antibody, anti-Ku80 antibody (ii,iii), GL4 (A1), or isotype-matched mouse monoclonal antibodies or rabbit serum (shadow), and then analyzed for the expression of P antigen or Ku80 on the cell surface. Figures show HeLa-mock expressed P antigen but not Ku antigen on the surface (i,ii), whereas HeLa-Ku80 expressed Ku80 (iii). (B) Increased binding and viral entry of B19 in HeLa-Ku80. The indicated cells (6 × 105) were infected with B19 (2 × 1011 copies of B19 DNA) for 30 minutes on ice. After washing cells 3 times with PBS, pH 7.2, DNA was extracted from 2 × 105 cells. Remaining cells were further incubated for 30 minutes at 37°C. After washing cells 3 times with PBS, pH 4.5, a cytoplasmic and nuclear fraction was prepared, and then DNA was extracted from each fraction. Prepared DNA was subjected to a quantitative PCR to quantify B19 DNA. *P < .01 by Student t test. (C) B19 infection to HeLa-Ku80. HeLa-mock or HeLa-Ku80 cells (2 × 105) were infected with B19 (2 × 1011 copies of B19 DNA) for 30 minutes at 37°C. After being washed 3 times with PBS, cells were collected with 5 mM EDTA-PBS, pH 7.2, fixed with 4% paraformaldehyde and reacted with PAR3, followed by FITC-labeled anti–mouse IgG antibody as a secondary antibody. Thus prepared cells were then subjected to a confocal microscope analysis. The panel represents B19 entered into HeLa-Ku80. (D) Colocalization of rB19ECP and Ku80. HeLa-mock or HeLa-Ku80 (2 × 105) cells were incubated with biotinylated rB19ECP (1 μg/mL) in the presence of 5 μg/mL inhibitor antibody indicated for 30 minutes at 37°C. After being washed 3 times with PBS, pH 7.2, cells were collected with 5 mM EDTA-PBS, and rB19ECP or Ku80 was detected by confocal microscopy analysis. Ku80 was detected by anti-Ku80 antibody followed by TRITC-labeled anti–mouse IgG antibody as a secondary antibody. Detection of biotinylated rB19ECP was done by avidin-FITC as described in “Materials and methods.”

Transfection of Ku80 to HeLa cells. (A) Expression of Ku80 on HeLa cells transfected with pKu80. Ku80 cDNA was inserted to expression plasmid pcD and the resulted pKu80 was transfected to HeLa cells (HeLa-Ku80) using lipofectin. Empty pcD was used for a mock transfection (HeLa-mock). The transfected cells (2 × 105) were incubated with 5 μg/mL of each antibody, anti-Ku80 antibody (ii,iii), GL4 (A1), or isotype-matched mouse monoclonal antibodies or rabbit serum (shadow), and then analyzed for the expression of P antigen or Ku80 on the cell surface. Figures show HeLa-mock expressed P antigen but not Ku antigen on the surface (i,ii), whereas HeLa-Ku80 expressed Ku80 (iii). (B) Increased binding and viral entry of B19 in HeLa-Ku80. The indicated cells (6 × 105) were infected with B19 (2 × 1011 copies of B19 DNA) for 30 minutes on ice. After washing cells 3 times with PBS, pH 7.2, DNA was extracted from 2 × 105 cells. Remaining cells were further incubated for 30 minutes at 37°C. After washing cells 3 times with PBS, pH 4.5, a cytoplasmic and nuclear fraction was prepared, and then DNA was extracted from each fraction. Prepared DNA was subjected to a quantitative PCR to quantify B19 DNA. *P < .01 by Student t test. (C) B19 infection to HeLa-Ku80. HeLa-mock or HeLa-Ku80 cells (2 × 105) were infected with B19 (2 × 1011 copies of B19 DNA) for 30 minutes at 37°C. After being washed 3 times with PBS, cells were collected with 5 mM EDTA-PBS, pH 7.2, fixed with 4% paraformaldehyde and reacted with PAR3, followed by FITC-labeled anti–mouse IgG antibody as a secondary antibody. Thus prepared cells were then subjected to a confocal microscope analysis. The panel represents B19 entered into HeLa-Ku80. (D) Colocalization of rB19ECP and Ku80. HeLa-mock or HeLa-Ku80 (2 × 105) cells were incubated with biotinylated rB19ECP (1 μg/mL) in the presence of 5 μg/mL inhibitor antibody indicated for 30 minutes at 37°C. After being washed 3 times with PBS, pH 7.2, cells were collected with 5 mM EDTA-PBS, and rB19ECP or Ku80 was detected by confocal microscopy analysis. Ku80 was detected by anti-Ku80 antibody followed by TRITC-labeled anti–mouse IgG antibody as a secondary antibody. Detection of biotinylated rB19ECP was done by avidin-FITC as described in “Materials and methods.”

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