Fig. 4

C/EBPβ is a transcription activator in the promoter region of Clec7a gene. (A and B) Schematic representation of a series of 5’ unidirectional deletions of the 2000 bp Clec7a promoter region fused in frame to pcDNA3.1 luciferase reporter vector, and then the promoter activity of C/EBPβ was determined by measuring the fluorescence intensity. C/EBPβ binding elements in the promoter region of Clec7a were predicted using the transcription activator search software, and shown as cyan boxes (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with pcDNA3.1-GFP-NC group via unpaired Student’s t test. (C) Mutated C/EBPβ binding sites on Clec7a promoter-driven luciferase reporters were constructed, and then the luciferase activity was analyzed via one-way ANOVA followed by Tukey’s post hoc test (n = 3). ^***P < 0.001 compared with the pcDNA3.1-GFP-NC + psiPRO-Clec7a-p500 group. ^###P < 0.001 compared with the pcDNA3.1-GFP-NC + psiPRO-Clec7a-p500-DEL group. D The location of amplified fragments of three pairs of primers on Clec7a promoter region. E Chromatin immunoprecipitation (ChIP) analysis of C/EBPβ binding at the Clec7a promoter. The anti-C/EBPβ antibody or IgG control was used to pull down the DNA–protein complex from the control or LPS + ATP treated BV2 cells as indicated. Subsequently, immunoprecipitated DNA was determined by PCR and quantitative real-time PCR with three pairs of primers (n = 3). Data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used to analyze data among multiple groups. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the BV2-C/EBPβ antibody group