Figure 3. Non-functional Chk1 mutants fail to undergo activating phosphorylation. (A and B) Western blotting (anti-HA for Chk1, anti-tubulin as a loading control) for the indicated strains either mock irradiated, or irradiated with 150 J/m2 UV-C. Activating phosphorylation is seen as a retarded mobility on SDS-PAGE. Note that the ΔKA1 and Δ488–489 mutants show no detectable protein expression, but (C) show normal mRNA levels by northern blotting (28S rRNA is shown as a loading control). (D) The indicated Chk1 mutants were expressed in cells lacking the Chk1 S345 phosphatase Dis2, and either mock-treated or incubated with 0.01% MMS for 7 h. The non-functional alleles still fail to show activating phosphorylation.