Regulation of ELK-1 activity by SUMO-1 modification. (A) TREx-F3H3-K-Rta-shSUMO-1 BCBL-1 cells were treated with Dox for 48 hours. TCLs were analyzed by immunoblotting using anti-SUMO-1 antibody. (B to H) Two ELK-1 targeted genes, TARS2 (B) and NDUFB7 (C), showing SUMO-1 enrichment at the promoter region identified in our study and three genes, SNRPE (D), INO80B (E) and LYSMD1 (F), that have high quality ELK-1 binding sites identified in HeLa cells overlapping with our SUMO-1 enriched regions were chosen. Two genes, MCL-1 (G) and IRF-3 (H), with ELK-1 binding site at the promoter region showing no SUMO-1 enrichment were chosen as control. RNA samples derived from TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta shSUMO-1 BCBL-1 cells before and after 48 hours of Dox induction were subjected to reverse transcription (RT) reaction. Following the RT reaction, the ELK-1 target genes were amplified by qPCR using gene-specific primer sets. All reactions were run in triplicate and normalized against GAPDH.