Extended Data Fig. 8: Electrophoretic mobility shift assay reveals cooperative binding of p52-ETS1 on human POU2AF1 promoter sequence.
From: The transcription complex p52–ETS1 is essential for germinal center formation
(a)Murine Pou2af1 promoter sequence. The highlighted ~330 bp region denotes the promoter segment containing two ETS1 binding sites (marked in red). (b) Human POU2AF1 promoter sequence used for the EMSA assay. The highlighted ~330 bp region denotes the promoter segment containing two ETS1 binding sites (marked in red). (c) Sequence alignment analysis showing the ETS1 binding sites (site 1 in purple and site 2 in blue) in the Pou2af1 promoter sequence to be conserved among human and mice. (d) Recombinant p52 and ETS1 proteins (500 ng) were analysed for cooperative binding on the POU2AF1 promoter DNA-labelled probes (Cy5 labeled) with EMSA. Data are representative of two independent experiments. Lane 1: Free POU2AF1 promoter DNA-Cy5 labelled probe, Lane 2: Binding of ETS1 alone with the labelled probe, Lane 3: Binding of p52 alone with the labelled probe, Lane 4: Cooperative binding of p52 and ETS1 proteins with the labelled probe (marked by the top arrow), Lane 5: Cooperative binding of p52RMK>A mutant and ETS1 proteins with the labelled probe.
