Fig. 3. Delayed phosphorylation of Net1 in clb2^NLS-expressing cells. (A) clb2^NLS but not clb2^L303A exacerbates the temperature-sensitive growth phenotype of cdc15-2. Serial 10x dilutions of saturated cultures were spotted on YPD plates and incubated at the indicated temperatures for 3-4 days. (B) The synthetic effect of the clb2^NLS allele is recessive. The same experiment as in A was repeated with cdc15-2/cdc15-2 diploid cells expressing the indicated CLB2 alleles. (C) The kinetics of Net1 phosphorylation was analyzed in extracts from clb2^NLS clb1 and clb1 cells expressing NET1-Myc and PDS1-HA alleles. Cells were released from an -factor-induced G1 arrest at 25°C and aliquots taken every 10 minutes. Total cellular extracts were separated by a long migration on 8% SDS-polyacrylamide gels to detect Net1-Myc, and by 10% SDS-PAGE for Clb2, Pds1 and Cdc28. To better compare the Net1 migration profiles, clb2^NLS clb1 and clb1 extracts were run on the same gel. Clb2 and Pds1 were used as markers of cell cycle progression.