Figure 2.

(A) Total p27 levels decrease rapidly after release from the DFO block. Confluent SKNSH cells were subcultured into RPMI/10% FCS with 100 μm DFO and incubated. After 20 h the medium was replaced with new medium as RM. The cells were harvested at regular intervals beginning 15 min after aspirating the medium and adding hot SDS loading buffer. Westerns were performed as described and (total) p27 levels were determined. Thereafter, the blot was stripped and probed for B Actin. Each treatment was conducted in duplicate. (B) p27 accumulation occurs in cells subcultured in DFO at the same time as cyclin E accumulation. Confluent untreated SKNSH cells were subcultured into RPMI/10% FCS with or without 100 μm DFO. The cells were harvested at 10 h and 20 h by aspirating the medium and treating with hot SDS loading buffer. Westerns were performed as described. First the levels of cyclin A were determined. Following this the blot was probed for cyclin E, p27, and β-actin with the blot stripped after each probe. (C) Although it was unstable, some experiments showed that p27 kip is phosphorylated at thr187 following reversal from DFO. Confluent SKNSH cells were subcultured into RPMI/10% FCS with 100 μm DFO and incubated. The medium was replaced with RM and with or without DFO. The medium was aspirated at the times indicated in the figures and westerns were performed, for py187p27 levels.