Fig. 4.

BmKRP directly binds the 20E CRE in the BmKr-h1 promoter. A DNA pull-down experiment with nuclear proteins isolated from BmN cells. The oligonucleotide probes are shown in the top panel. WT, wild-type and MT, mutated ssDNA. The “*” point to the protein band that was observed in WT but not in the MT. B Schematic representation of the protein structure of BmKRP (Accession no. XM_004922171.3). C Subcellular localization of BmKRP protein in BmN cells. BmN cells were transfected with pEGFP (a–c) or BmKRP-EGFP (d–f). The bars represent 15 μm. N, nucleus; C, cytoplasm. D EMSA of translated recombinant BmKRP protein binding to the − 248 ~ − 217 nt probe. E Chromatin immunoprecipitation (ChIP) assay of expressed BmKRP-EGFP binding to the 20E CRE in the BmKr-h1 promoter in BmN cells. The ChIP target sequence was detected by qRT-PCR. The enrichment of the promoter sequence in the immunoprecipitated DNA samples was normalized with DNA present in the 10% input material. F The sequence of the − 282 ~ − 181 nt region of the BmKr-h1 promoter. The region that bound to BmKRP is boxed in red. The primer aligned regions are underlined. G The sequencing atlas of the enriched RT-PCR product of the ChIP assay. H Effect of BmKRP on luciferase activity driven by the BmKr-h1 promoter with either the wild-type or mutant form of the 20E CRE in BmN cells. I BmKRP expression detected at 48 h after BmKRP dsRNA was transfected into BmN cells. J Changes in luciferase activity in BmN cells co-transfected with BmKRP dsRNA or EGFP dsRNA and the luciferase reporter vector treated by 20E. The data in E, H, I, and J are means ± SEM (n = 3), and the individual data values are shown in Additional file 2. Different letters above the columns indicate significant differences in luminescence at p < 0.05 by ANOVA. The significance of the differences between the treatment and control was statistically analyzed at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) using t test