Fig. 6.

SUMO represses global transcription via CDK9 sumoylation. a Generation of HEK293T cell lines with knockout of endogenous CDK9 and expression of Myc-CDK9 or SUMO-deficient Myc-CDK9K/R mutant. The lack of endogenous CDK9 and expression of Myc-CDK9 or Myc-CDK9K/R mutant were verified by WB analysis using anti-CDK9 antibody. The cellular extracts were immunoprecipitated with anti-Myc antibody, followed by WB analysis using either Myc or SUMO-1 antibody as indicated. b WB analysis showing that ectopic expression of PIAS1/SUMO-1 inhibited Ser2P in CDK9KO/Myc-CDK9 cells but not in CDK9KO/Myc-CDK9K/R cells. c The cellular extracts in (b) were immunoprecipitated with anti-Myc antibody and the half of the resulting proteins were analyzed by WB using anti-Myc or anti-Cyc T1. The rest of half samples were used for in vitro kinase assay using GST-CTD as substrate and the levels of Ser2P were detected by WB analysis. d The measurement of total RNAs from equivalent numbers of cells showing that ectopic expression of PIAS1/SUMO-1 inhibited global transcription in CDK9KO/Myc-CDK9 cells but not in CDK9KO/Myc-CDK9K/R cells. e RT-PCR analysis of representative SUMO-regulated genes showing that ectopic expression of PIAS1/SUMO-1 inhibited transcription in CDK9KO/Myc-CDK9 cells but not in CDK9KO/Myc-CDK9K/R cells. f IF staining showing that ectopic expression of PIAS1/SUMO-1 reduced the levels of Ser2P in the CDK9KO/Myc-CDK9 but not in the CDK9KO/Myc-CDK9K/R cells. g BrU incorporation assay showing that ectopic expression of PIAS1/SUMO-1 substantially reduced the levels of BrU incorporation in CDK9KO/Myc-CDK9 but not in CDK9KO/Myc-CDK9K/R cells