Figure 6. Phosphorylation on T584 promotes cell cycle–dependent phosphorylation of both T275 and S278. (A) Antibodies raised against a peptide corresponding with Sid4 simultaneously phosphorylated on T275 and S278 used to blot Sid4 preparations immunoprecipitated from denatured extracts of the indicated strains. Immunoprecipitates were incubated for 30 min at 30°C with λ phosphatase, its inhibitor, or buffer alone, as indicated. (B–D) Small G2 dma1.Δ (B), sid4.GFP ppc89.GBP dma1.Δ (C), or sid4.T584EGFP ppc89.GBP dma1.Δ (D) cells were isolated from mid-log phase cultures by centrifugal elutriation at t = 0, and aliquots were taken at the indicated intervals to monitor septation and new end take off (NETO) with calcofluor white or phosphorylation on both T275 and S278. Control samples 40 min after cdc25.22 dma1.Δ control cultures were released from 4.25 h arrest at 36°C as indicated.
Figure 6.

Phosphorylation on T584 promotes cell cycle–dependent phosphorylation of both T275 and S278. (A) Antibodies raised against a peptide corresponding with Sid4 simultaneously phosphorylated on T275 and S278 used to blot Sid4 preparations immunoprecipitated from denatured extracts of the indicated strains. Immunoprecipitates were incubated for 30 min at 30°C with λ phosphatase, its inhibitor, or buffer alone, as indicated. (B–D) Small G2 dma1.Δ (B), sid4.GFP ppc89.GBP dma1.Δ (C), or sid4.T584EGFP ppc89.GBP dma1.Δ (D) cells were isolated from mid-log phase cultures by centrifugal elutriation at t = 0, and aliquots were taken at the indicated intervals to monitor septation and new end take off (NETO) with calcofluor white or phosphorylation on both T275 and S278. Control samples 40 min after cdc25.22 dma1.Δ control cultures were released from 4.25 h arrest at 36°C as indicated.

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