Band depletion does not reflect destruction of topo I. HL-60 cells were treated with diluent (lanes 1 through 4) or 10 μmol/L TPT for 45 minutes at 37°C (lanes 5 through 8) and sedimented at 3,200g for 1 minute. All of the supernatant except for 50 μL was removed. Cells were resuspended in the remaining 50 μL of supernatant and heated to 48°C for 0 minutes (lanes 1 through 5), 1 minute (lane 6), 2 minutes (lane 7), or 3 minutes (lane 8) before addition of lysis buffer. After preparation for electrophoresis as described in the Materials and Methods, samples containing polypeptides from 3 × 105 cells (lanes 1 and 5 through 8) or the serial dilution described in the legend to Fig 1 (lanes 2 through 4) were loaded in adjacent lanes. Blots were probed with antibodies to topo I (A) or poly(ADP-ribose) polymerase (B).
