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. 2006 Nov 27;27(3):1158–1171. doi: 10.1128/MCB.01745-05

FIG. 3.

FIG. 3.

(A) Lethality of Cdc6 phosphorylation site mutants in a mih1Δ background. W303 or isogenic mih1Δ (W303 mih1Δ) strains expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector were grown in glucose-containing selective medium to stationary phase, serially diluted 10-fold, spotted in either raffinose (as a control) or raffinose plus galactose selective plates, and incubated at 30°C for 2 days. The mih1Δ strains were YSB198, empty vector; YSB199, GAL-CDC6; YSB157, GAL-CDC6 E+F; YSB201, GAL-CDC6 A-C+F; YSB202, GAL-CDC6 A-F; and YSB203, GAL-CDC6 ΔN8-48. (B) Interaction between Cdc6p and Cdc28p. Strains (YSB161, empty vector; YSB162, GAL-CDC6; YSB163, GAL-Cdc6 F; YSB164, GAL-CDC6 A-C; YSB166, GAL-CDC6 A-C+F; and YSB167, GAL-CDC6 A-F) containing HA-tagged Cdc28p under the control of its natural promoter and expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector were grown in YPR to mid-log phase, and galactose was added to induce the expression of Cdc6p. After 2 h, total yeast extracts were prepared and subjected to immunoprecipitation (IP). The top panel corresponds to HA immunoprecipitates blotted with the 9E10 anti-myc antibody. The bottom panel corresponds to myc immunoprecipitates blotted with the 12CA5 anti-HA antibody. (C) Yeast cells with a copy of MET-CDC6 and another copy of myc-tagged wild-type CDC6 (YSB75) or CDC6 A-C (YSB133) expressed under its own promoter and containing a chromosomal copy of Pds1-HA or Clb2-HA were grown in synthetic medium without methionine and arrested with α-factor (the Pds1-HA strains were YSB441 and YSB478, and the Clb2-HA strains were YSB407 and YSB472). The cells were further treated as described in the legend for Fig. 1B. (D) Effect of wild-type and mutant Cdc6p overexpression on Cdc28 kinase activity. W303 strains (YSB359, empty vector; YSB360, GAL-CDC6; YSB361, GAL-CDC6 F; and YSB364, GAL-CDC6 A-C+F) containing HA-tagged p86/91 and expressing the indicated forms of myc-tagged Cdc6p under the GAL1,10 promoter were grown to early log phase in YPRaf and arrested with nocodazole (Noc). After nocodazole arrest, galactose was added for 2.5 h and samples were taken to be processed for Western blotting to detect p86/91. wt, wild type.