Figure 5.
Immunoprecipitation of CDK8 and Thermal Stability Assay
(A) Lysates from HEK293T cells transiently transfected with Myc-FLAG-tagged wild-type (WT) or mutant CDK8 constructs were used for immunoprecipitation with anti-FLAG antibody or mouse IgG isotype control antibody. The immunocomplexes were purified with protein G magnetic beads. CDK8 was released by adding FLAG peptide, and an aliquot of the eluted fraction was saved for immunoblot analysis with anti-Myc or anti-cyclin C (CycC) antibodies.
(B) A thermal stability assay of the eluted fractions incubated in the absence or presence of ATP and heated individually at different temperatures (gradient between 50–82°C). Virtual blot views of results for WT and the mutants are shown. For each CDK8 construct, the red arrowhead indicates 50% reduction of the CDK8 signal compared to the signal at the lowest temperature.