Fig 1: Interaction between Zkscan3 and the GATA1 promoter. A We constructed the Zkscan3-His-GFP fusion insert, and transfected CT26 cells using lentivirus carrying the fusion insert. Quantitative PCR was performed to detect Zkscan3 expression in CT26 cells (n = 3, Student’s t test). B We cloned the − 1000–0 bp Gata1 promoter sequence into the pGL4.17 vector and co-transfected CT26 cells overexpressing Zkscan3 and CT26 cells using the Renilla luciferase plasmid. Luciferase activity was determined 48 h after transfection using a Dual-Luciferase assay kit (Promega, Madison, WI, USA) and measured with a microplate luminometer (Thermo Fisher Scientific). Luciferase reporter assay showing the effect of Zkscan3 on GATA1 promoter activity (n = 3, Student’s t test). (C) Nuclear extract of CT26-ZK3 cells was used to perform co-immunoprecipitation followed by western blot analysis. (D) We transfected MEL cells with the lentivirus carrying Zkscan3-His-GFP fusion insert, and detected the GFP expression by flow cytometry (n = 3, Student’s t test). (E) Nuclear extract of MEL-ZK3 cells was used to perform co-immunoprecipitation followed by western blot analysis. For detailed experimental procedures, please see “Materials and methods” section. F Chromatin immunoprecipitation (ChIP) of GATA1 promoter with ZKSCAN3 in Zkscan3-overexpressing MEL cells (MEL-Zkscan3). His-tagged ZKSCAN3 protein was pulled down using a specific anti-His antibody. To detect the binding of ZKSCAN3 to the GATA1 promoter, cross-linked DNA was amplified using quantitative PCR and specific primers. The positive control and the negative control antibodies and primers were provided in the kit. All experiments were performed according to manufacturer’s instructions (catalog number: 56383, Cell Signaling Technology) (n = 3, Student’s t test). (***p < 0.001, **p < 0.01, *p < 0.05)