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. 2012 Sep 1;23(17):3485–3497. doi: 10.1091/mbc.E12-03-0231

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© 2012 Zeng 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 6: — Cdc28–Cln3 phosphorylation of Sla1 enhances its phosphorylation by Prk1 and reduces its association with Pan1. (A) Detection of Prk1-mediated phosphorylation of Sla1. Yeast cells of GZY652 (SLA1-WT), GZY658 (sla1-13A), and GZY670 (sla1-13E) were incubated for hyphal growth with 20% FBS at 37°C for 2 h. Sla1 (tagged with HA) was immunoprecipitated with αHA and subjected to λPP or mock treatment. For WB analysis, the membrane was first probed with αP-Thr and then stripped for reprobing with αHA. (B) Comparison of Prk1-mediated phosphorylation on Sla1-13A and Sla1-13E in A. The phosphorylation intensity of Sla1-WT-HA, Sla1-13A-HA, and Sla1-13E-HA (detected by αP-Thr) was measured by a densitometer and normalized against the amount of respective protein (detected by αHA). The results were then calculated as percentages against the normalized value of Sla1-WT-HA. (C) CoIP of Sla1-WT, Sla-13A, and Sla-13E with Pan1. Yeast cells of GZY718 (SLA1-WT-HA PAN1-Myc), GZY719 (sla1-13A-HA PAN1-Myc), and GZY720 (sla1-13E-HA PAN1-Myc) were incubated with 20% FBS at 37°C for 2 h. Cell extracts were prepared and subjected to αMyc IP, followed by immunoblotting with αMyc and αHA. (D) Comparison of the amounts of Sla1-13A and Sla1-13E coimmunoprecipitated by Pan1 in C. The amount of Sla1-WT-HA, Sla1-13A-HA, and Sla1-13E-HA was measured by a densitometer and normalized against the amount of Pan1-Myc used for coIP, respectively. The results were then calculated as percentages against the normalized value of Sla1-WT-HA. (E) CoIP of Pan1 with Sla1-WT, Sla1-13A, and Sla1-13E. Cells of GZY718, GZY719, and GZY720 were incubated with 20% FBS at 37°C for 2 h. Cell extracts were prepared and subjected to αHA IP, followed by WB with αHA and αMyc. (F) Comparison of the amounts of Pan1 coimmunoprecipitated with Sla1-13A-HA and Sla1-13E-HA in E. The amount of Pan1-Myc was measured by a densitometer and normalized against the amount of Sla1-WT-HA, Sla1-13A-HA, and Sla1-13E-HA used for coIP, respectively. The results were then calculated as percentages against the value for Sla1-WT-HA.