Fig 4.

Analysis of Sum1-3Flag, Hst1-5HA, and Ndt80 proteins in various cdc7-as strains without and with PP1. (A) A cdc7-as SUM1-3Flag HST1-5HA diploid (NH1078) was transferred to Spo medium in the absence or presence of 15 μM PP1, and total cell extracts were prepared at the indicated time points. Sum1-3Flag, Hst1-5HA, and Ndt80 were analyzed using immunoblot assays with anti-Flag, anti-HA, and anti-Ndt80 antibodies, respectively. (B) A cdc7-as ndt80Δ SUM1-3Flag diploid (NH1068) was transferred to Spo medium and incubated for 8 h without or with 15 μM PP1. Sum1-3Flag was detected on immunoblots using anti-Flag antibodies. (C) Sum1-3Flag protein was immunoprecipitated from NH1068 incubated after transfer to Spo medium for 0 h, 8 h, or 8 h with 15 μM PP1. Half of the immunoprecipitates were treated with λ protein phosphatase as indicated and then probed with anti-Flag antibodies.