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. 2014 Feb 3;111(7):2500–2505. doi: 10.1073/pnas.1307525111

Fig. 4.

Fig. 4.

S191 in Med3 is required for interaction with Grr1 and loss of Med3 stability. Med3 stability is increased following mutation on S191. (A) WT cells transformed with MED3 or MED3 (S191G) or MED3 (T195A) together with CDK8 or CDK8 (D290A). Total protein was extracted from cells, and following separation, immunoblots were probed with anti-HA for Med3. Med3 interacts with Grr1. (B) Yeast two-hybrid assay using pASGrr1 and HA-Med3 (WT or S191G/T195A variants) in the presence of Cdk8 or Cdk8 (D290A). Cells were grown to midlog phase and assayed for LacZ expression. pACTII, negative control. (C) Phosphorylation of S191 in Med3 is needed for Med3-Grr1 interaction. Biotin-labeled Grr1 was incubated with Med3 immuno-precipitated from cells expressing HA-Med3 (WT or S191G/T195A variants) and cdk8. Grr1 was visualized using streptavidin-HRP and Med3 using αHA-antibody and chemiluminescent detection. (D) Med3(S191G) cannot repress FLO11 expression. Cells were transformed with MED3 (WT or S191G/T195A variants), CDK8 or Cdk8 (D290A), and GRR1 (where indicated). Total RNA was isolated and FLO11 expression levels determined using qRT-PCR. Values are MED3 set at 100% and normalized to ACT1.