Supplementary Figure 4: Cyclin C-CDK8, C-CDK19 and C-CDK3 kinases regulate ICN1.

(a) Protein sequences for human CDK8 (GI: 4502745), CDK19 (GI: 30387611), and CDK3 (GI: 4557439) were obtained from NCBI RefSeq. Multiple sequence alignment was performed with MAFFT (version 7) using the L-INS-i method. The resulting alignment was visualized using Jalview 2.8, highlighting identical amino acids between all three kinases in dark blue, and identical amino acids between two of the three kinases in light blue. (b–d) MOLT-16 cells were transduced with viruses encoding shRNAs against the indicated CDKs. The levels of ICN1 and CDKs were then determined by immunoblotting. (e) Validation of anti-CDK3 shRNAs. HeLa cells stably expressing HA-tagged CDK3 were used (to allow unequivocal detection of CDK3 with anti-HA antibody). Cells were transduced with viruses expressing five different anti-CDK3 shRNAs (1-5). Knockdown of CDK3 was gauged by immunoblotting with an anti-HA antibody. shRNAs #2 and #3 were chosen and used for analyses shown in d (f) His-tagged CDK19, or cyclin C, or cyclin C plus His-tagged CDK19, were expressed in 293T cells. Complexes were immunoprecipitated using anti-His antibody (or with IgG, for control) and used in in vitro kinase reactions in the presence (+) or absence (−) of ICN1 as a substrate together with γ[³²P]ATP. As a positive control, ICN1 was incubated with recombinant cyclin-C-CDK8. Upper panel: proteins analyzed by autoradiography to detect phosphorylated ICN1 (³²P-ICN1). Second panel: total GST-ICN1 protein detected by immunoblotting with anti-GST antibody (ICN1). Third and fourth panels: CDK19 and cyclin C detected using the indicated antibodies (anti-His antibody was used to detect tagged CDK19). (g) Whole cell lysates (Input) from experiment shown in f, immunoblotted with the indicated antibodies. Anti-His antibody was used to detect tagged CDK19. (h) HA-tagged CDK3, or cyclin C, or cyclin C plus HA-tagged CDK3 were expressed in 293T cells. Complexes were immunoprecipitated using anti-HA antibody (or with IgG, for control), and used in in vitro kinase reactions in the presence (+) or absence (−) of ICN1 as a substrate together with γ[³²P]ATP. As a positive control, ICN1 was incubated with recombinant cyclin C-CDK8. Upper panel: proteins analyzed by autoradiography to detect phosphorylated ICN1 (³²P-ICN1). Second panel: total GST-ICN1 proteins detected by Ponceau S staining. Third and fourth panels: CDK3 and cyclin C were detected using the indicated antibodies (anti-HA antibody was used to detect tagged CDK3). (i) Whole cell lysates (Input) from experiment shown in h, immunoblotted with the indicated antibodies. Anti-HA antibody was used to detect tagged CDK3.