Figure 3

Phosphorylation of Net1 is modulated by Cdc5.  NET1-myc9 (+, Lanes 1-4) in the wild-type ("+", RJD1349), cdc5-1 ("5", RJD1417), cdc14-1 ("14", RJD1408) and cdc5-1 cdc14-1 ("5, 14", WY334) backgrounds or net1(7m: S30, S31, S48, S60, S64, S242, S335 -> A)-myc9 (7m, Lanes 5-8) cells in corresponding backgrounds ("+", WY347; "5", WY363; "14", WY351; "5, 14", WY378) were grown in YP + 2% glucose + 1 M sorbitol at 25°C (cdc5-1 cdc14-1 cells seemed to grow more consistently in sorbitol-containing media), and shifted to 37°C for 3 hours when > 90% of cdc5, cdc14, and cdc5 cdc14 cells were arrested at the large-bud stage. Cell extracts were prepared by SDS-boiling method [8], fractionated on a 7.5% SDS-polyacrylamide gel, and Net1-Myc9 was detected by immunoblotting with 9E10 antibodies. The reduced mobility of Net1 isolated from cdc14-1 compared to wild type cells is due to phosphorylation [8].