Figure 3.
CTD Ser-5 hypophosphorylation without CAK-deficiency in mcs6 and pmh1 mutants. In the indicated strains, incubated at 36°C for indicated times, we measured levels of wild-type and mutant, HA-tagged Pmh1, and Mcs6 proteins by immunoblotting with anti-HA antibody (A, top) and Pmh1- or Mcs6-associated CTD kinase in anti-HA immunoprecipitates (A, bottom); CTD Ser-5 phosphorylation (B, top) and total Pol II (B, bottom) by immunoblotting of total yeast extracts with antibodies H14 and 8WG16, respectively; and Cdc2 Thr-167 phosphorylation, by immunoblotting of p13Suc1 precipitates (C). As a control, Cdc2 Thr-167 phosphorylation in the CAK-deficient, mcs6-13 csk1Δ strain was analyzed at 28°C and at 6 h after shift to 36°C.