Lims1 is a direct transcriptional target of NF-E2. (A) Schema of reporter constructs with presence or deletion of putative NF-E2 binding sites, which are indicated in terms of their position relative to the transcription start site (+1). (B) Results of luciferase reporter assays for the Lims1 promoter region and its deletions variants. COS cells were transfected with the reporter (pGL3 or LIM series) construct in the presence or absence of p45 NF-E2 expression plasmid. (C) Quantitation of MK chromatin IP (ChIP) results for the 2 putative NF-E2 binding sites in the Lims1 promoter and controls. Immunoprecipitated DNA was amplified by qPCR and the abundance of individual fragments is normalized to that of input (pre-IP) DNA. LIM-495 and LIM-1939 amplify corresponding fragments in the Lims1 promoter and LIM-down interrogates sequences approximately 4 kb downstream; other controls test for IP of promoter fragments in the Apo1a and HistoneH4a genes. (D) Agarose gel electrophoresis of PCR fragments after ChIP. The signals for input DNA with all primer sets and for LIM-1939 after NF-E2 IP were saturated well before the 33 cycles of PCR applied in this experiment; all other signals seem to represent background. Error bars in (B) represent standard deviation.