GPS2 interacts with EKLF and enhances EKLF-mediated transcriptional activity. (A-E) Luciferase reporter assays to test the effects of GPS2 on EKLF- and GATA1-mediated transcriptional activity. HEK293T cells were transfected with various combinations of plasmids as indicated below each column. Relative luciferase activity was measured after 48 hours by a dual-luciferase reporter assay. All activities were normalized to pGL3-Basic activity. Overexpression of GPS2 promotes EKLF-induced (A), but not GATA1-induced (B), luciferase activity of ALAS2-pro-luc plasmid and increases EKLF-induced luciferase activity of AHSP-pro-luc (C) and HBB-pro-luc plasmid (D). Disruption of the EKLF-binding site in the ALAS2-pro-luc reporter construct obviously weakened the promotion of EKLF-induced luciferase activity by GPS2 overexpression (E). All values are mean ± SEM (n = 3 replicates). (F) HEK293T cells were transfected with Myc-EKLF together with Flag-GPS2 or control plasmid. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated (IP) with anti-Flag M2 agarose. (G) Lysates of MEL cells were immunoprecipitated with GPS2 antibody or normal rabbit immunoglobulin G (IgG) and then subjected to immunoblot with EKLF or GPS2 antibody. (H) A schematic representation of EKLF WT and deletion mutants. (I) HEK293T cells were transfected with various combinations of plasmids encoding Flag-GPS2 and Myc-EKLF or EKLF deletion mutants as indicated. Cell lysates were pulled down with anti-c-Myc agarose and subjected to immunoblot with Myc or Flag antibody. (J) A schematic representation of GPS2 WT and deletion mutants. (K) HEK293T cells were transfected with various combinations of plasmids encoding Myc-EKLF and Flag-GPS2 or GPS2 deletion mutants as indicated. Cell lysates were pulled down with anti-Flag M2 agarose and subjected to immunoblot with Myc or Flag antibody. Data are representative of three independent experiments (F-G,I,K). ***P < .001; 2-tailed unpaired t test.