Figure 6. CDK12 expression and Immuno-purified CDK12 kinase assays from the parental and CDK12^as cell lines. (a.) Western blots of equal numbers of parental (WT) and CDK12^as cells using an anti-CDK12 antibody; Rpb2 is used as a loading control. CDK12 protein levels in the CDK12^as cell line are quantified to be ~50% to that of the parental cell line (Below image; Licor Odyssey software). The anti-CDK12 antibody targets the N-terminal arm of CDK12 (amino acids 201-220), well before the stop codon near amino acid 813. (b.) CDK12 was purified using anti-CDK12 antibody saturated protein G dynabeads from nuclear extracts of both the parental (WT) and analog sensitive (CDK12^as) cell line. CTD kinase activity was assayed using 1 μg of GST-yCTD fusion protein at 30 μM ATP, 37 °C, and a 30 min reaction time in the presence of [γ³²-P]ATP and DMSO or the bulky adenine analogue 1-NM-PP1 at 2 μM; the reactions were analyzed by SDS-PAGE and visualized using a PhosphorImager. Equal amounts of nuclear extract and dynabeads were used for each pulldown and reaction. It is worth noting that since equal amounts of cells were used as input in the reaction, we expect the amount of CDK12^as in the reaction to be half that of WT (see a.).