Mutagenic mimicry of phosphorylation at T584 imparts a sin⁻ phenotype. (A) A scheme showing the configuration of sid4.T584E strains. (B) Representative images of tubulin immunofluorescence as in Fig. 1 A of sid4.T584E cells 3 h after an early log phase culture was shifted from 19°C to 36°C. The binucleated interphase cells, indicated by arrows, arise from cytokinesis/septation failure in the previous cell cycle because of abolition of SIN function. n = 3. See also Fig. S2 B. BF, brightfield. (C) Spot tests of the indicated strains after growth on minimal (EMM2, no thiamine sid4⁺ expressed), or rich (YES) medium (contains thiamine to repress sid4⁺) at the indicated temperatures. (D) Representative monopolar counts as in Fig. 1 (C and D). See also Fig. S1 B. n = 3. (E–H) Western blots exploiting polyclonal antibodies that recognize Sid4 when phosphorylated on either T584 or simultaneously on both T275 and S278, all species of either Sid4 or Fin1, or the myc epitope tags on Pom1, as indicated. (E) Blots of Sid4 immunoprecipitates of denatured samples from cdc25.22 dma1.Δ cultures returned to 25°C 4.25 h after a shift to 36°C. As T584 phosphorylation peaks 40 min after release (not depicted), the impact of compromising kinase activities upon T584 phosphorylation was monitored at this time point in the figure. (F) Fin1 and Sid2 were isolated with polyclonal antibodies, whereas Pom1 was isolated with antibodies against the myc epitope, for kinase assays to identify which kinase could directly phosphorylate recombinant full-length Sid4 purified from E. coli. (G) Polyclonal antibodies precipitated Fin1 from asynchronous cultures of the indicated strains for in vitro kinase assays with recombinant Sid4. T584 phosphorylation was detected with the antibodies used in E.