c-Myb occupancy on the MYC promoter is SUMO-independent. ChIP were performed with K562 cells to assess occupancy of c-Myb wild-type and SUMO-negative 2KR-mutant on the human MYC promoter. K562 cells were stably transfected with 3×FLAG- and HA-tagged c-Myb wild-type and 2KR, and clones were picked based on immunoblotting (inserted panel) showing similar expression of the integrated and endogenous c-Myb. ChIP was performed using anti-FLAG antibody, while an isotype IgG antibody was used as negative control. Occupancy was analysed by amplifying the MYC promoter by real-time PCR after reversal of the cross-linking. An unrelated DNA region (UDR) was used as negative control. The UDR was chosen from the gene desert region (53), the exact location is: chr2: 22153688+22153788. The results are calculated from triplicates of real-time PCR reactions and are expressed as percentage of recovery ± SD relative to the input. v, stable cell lines with integrated empty vector pEF1neo; asterisk, endogenous c-Myb; double asterisk, stably integrated, double-tagged c-Myb.
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