Journal: Cell & Bioscience
Article Title: IgD in nucleus of pro-B cells promotes pro-B cells proliferation by regulating E2F3 expression
doi: 10.1186/s13578-025-01490-y
Figure Lengend Snippet: IgD deletion reduces the accessibility of E2f3a gene to inhibit E2f3a expression. A A heatmap representation of ChIP-seq data of transcription factors (TFs) was utilized to evaluate the genomic occupancy of IgD. B Genome browser tracks illustrate the promoter region at the E2f3 target site in pro-B cells, comparing conditions with anti-IgD to those with an isotype IgG control. C EMSA was used to confirm the specific binding of IgD to the E2f3 promoter region, with biotin-labeled sequences of the E2f3 promoter region used as probes. The assay included a non-biotinylated competition probe (Com Probe), representing an unlabeled sequence of the E2f3 promoter, and a mutation probe (Mut Probe) which has identical sequence with targeted nucleotide substitutions in the core E2F binding motif. The reaction system is depicted in the figure. D Supershift assay detects the specific binding of IgD to the E2f3 promoter region, the reaction system is as shown in the figure. E ChIP-PCR was performed to amplify the promoter regions of E2f3a and E2f3b using pro-B cells from WT mice, with genomic DNA from pro-B cells serving as a positive control. F Transcriptional levels of E2F3a and E2F3b in pro-B cell and pre-B cell derived from IgD −/− mice were detected by RT-PCR. G Protein expression levels of E2f3a in pro-B cell from WT and IgD −/− mice were examined by Western blot analysis. Quantification result was presented as grey scale scans with statistical significance denoted by asterisks (**** p < 0.0001)
Article Snippet: Primary antibodies used included anti-mouse β-actin (Zhongshan Golden Bridge, TA-09), anti-mouse Gapdh (Zhongshan Golden Bridge, TA-08), anti-mouse IgD (Santa, sc-53853), anti-mouse IgM (Bethyl, A90-101P), anti-mouse Igκ (SouthernBiotech, 1050-08), and anti-mouse E2f3 (CST, DF12390).
Techniques: Expressing, ChIP-sequencing, Control, Binding Assay, Labeling, Sequencing, Mutagenesis, Positive Control, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot