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. 2011 Mar 7;108(12):4926–4931. doi: 10.1073/pnas.1010010108

Fig. 3.

Fig. 3.

Maf1 is phosphorylated by CK2. (A) CK2 phosphorylates recombinant S. cerevisiae Maf1 expressed in E. coli and purified by HIS-tag isolation. Autoradiogram shows [γ32P]-ATP–labeled products of reactions containing 100 ng yeast TAP-tag purified CK2 and 1 μg casein (lane 0); increasing amounts of recombinant yeast Maf1 (0, 0.25, 0.5, 1, and 2 μg; lanes 1–5); 2 μg recombinant yeast Maf1 and 4% (vol/vol) DMSO (lane 6); 2 μg recombinant yeast Maf1, 4% (vol/vol) DMSO and 10 μM TBBt (lane 7). (B) Mutations of CK2 sites prevent Maf1 phosphorylation in vivo. Substitutions in MAF1 cloned in pRS315 vector were made using mutagenic PCR. Yeast mutants 1StA (S388A), 4StA (S159A, S160A, S161A, and S162A), 5StA (S159A, S160A, S161A, S162A, and S388A) and corresponding WT were grown overnight in SC –leu medium, then in glucose-rich medium to exponential phase (YPD Exp), transferred to glycerol medium for 3 h (YPGly), and again transferred to YPD for 3 h. TCA-precipitated proteins were examined by Western blot using anti-Maf1 antibody.