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. 2011 Oct 1;22(19):3595–3608. doi: 10.1091/mbc.E11-04-0340

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© 2011 Harvey et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Phosphorylation of Cdc28 consensus sites is necessary for Swe1 activity in vivo. (A) Purified Cdc28/Clb2 quantitatively phosphorylates purified Swe1 in vitro. Phosphorylation of Swe1 causes an electrophoretic mobility shift that is detected by Western blot analysis. (B) Wild-type and swe1-8A cells were released from a G1 arrest, and the percentage of cells with short mitotic spindles was determined at the indicated times and plotted as a function of time. Error bars represent SEM for three independent experiments. (C) Cell size distributions of log-phase cultures of wild-type, swe1Δ, and swe1-8A strains. Each trace is the average of 15 independent cultures. (D) Cells of the indicated genotypes were released from a G1 arrest, and samples were taken at the indicated times. Cdc28 phosphotyrosine was monitored by Western blotting. (E) Wild-type and swe1-8A cells carrying GAL1-3×HA-CLB2 were grown to midlog phase, and expression of 3×HA-Clb2 was induced with 2% galactose for 3 h at 30°C. Extracts were made, and 3×HA-Clb2 was immunoprecipitated with an anti-HA antibody. As a control, identical precipitations were carried out using an anti-GST antibody. Coprecipitation of Swe1 was assayed by Western blotting. The panels labeled “Extract” show Western blots of 3×HA-Clb2 and Swe1 in the crude extracts used for the immunoprecipitations. (F) Purified 3×HA-Swe1, 3×HA-swe1-8A, or 3×HA-swe1-10ncs were incubated with increasing amounts of purified cdc28-Y19F/Clb2-3×HA in the presence of ATP. Phosphorylation of Swe1 was detected as an electrophoretic mobility shift on a Western blot.