Fig. 2.

Cellular CDK9 is abundantly sumoylated and this correlates with suppression of Ser2 phosphorylaiton. a WB analysis showing that ectopically expressed CDK9 was extensively sumoylated when co-expressed with PIAS1/SUMO-1 in HEK293T cells. b Interaction between CDK9 and PIAS1 ectopically expressed in HEK293T cells determined by co-immunoprecipitation assay. c Interaction between CDK9 and UBC9 ectopically expressed in HEK293T cells revealed by co-immunoprecipitation assay. d In vitro sumoylation assay showing that recombinant CDK9 proteins were sumoylated in the presence of SUMO E1, E2 and 6x His-SUMO proteins. e WB analysis of 293T cell extracts showing the presence of 42 kDa and multiple higher molecular weight CDK9 bands that were all substantially reduced upon knockdown of CDK9 by two different siRNAs. f, g Knockdown of UBC9 in 293T (f) and HeLa and HCT116 (g) by two different UBC9 shRNAs resulted in marked reduction of the higher molecular-mass sumoylated CDK9 bands and the increased levels of Ser2P . h WB analysis showing increased higher molecular-mass sumoylated CDK9 bands in Senp1^−/− and Senp2^−/− MEF cells in comparison to the wild-type MEFs. i WB analysis of stable Myc-CDK9 HeLa cell extracts showing the presence of higher molecular-weight forms of Myc-CDK9. Knockdown of UBC9 by two different shRNAs resulted in substantial reduction of the higher molecular-weight forms of Myc-CDK9. j WB analysis of HeLa cell extracts stably expressing Myc-CDK9K/R or Myc-CDK9 and transfected with PIAS1 or PIAS1/SUMO-1 as indicated. No sumoylation was detected for the Myc-CDK9K/R mutant