Figure 3.

Chk1 kinase is activated in rid mutants when grown at the critical temperature, and Chk1 activation is not dependent on expression of mrc1. (A) Phosphorylation of HA-tagged Chk1-protein was detected by Western immunoblotting using anti-HA antibodies. On activation, Chk1 shows a characteristic mobility shift as indicated by the arrow. The rid mutants cdc20, cdc30, and cdc18 containing a HA-tagged version of Chk1 were shifted to their respective critical temperatures for 3 h and assayed for Chk1 activation. Chk1 is activated in all three rid mutant strains. (B) Mrc1 is not required for Chk1 activation in rid mutants. When shifted to the critical temperature of 32°C, Chk1 phosphorylation is observed in the rid mutant cdc20Δmrc1 (lane 4). In contrast, no Chk1 phosphorylation is observed in the strain cdc20Δcrb2 (lane 6).