Disrupted Aid expression and enzymatic activity in Aid knockout cells. (A) Whole BM cells were collected from WT, Aid^+/−, and Aid^−/− adult mice around 8 weeks old. Total RNA was extracted from these cells and mRNA expression of Aid and Gapdh was confirmed by semiquantitative RT-PCR. (B) mRNA expression levels of Aid were measured by RT-PCR using BM cells from WT, Aid^+/−, and Aid^−/− adult mice. Data were analyzed by the Δ cycle threshold (Ct) ratio technique using the murine Gapdh gene as a housekeeping gene. Results are presented as the ratio of the Aid^+/− or Aid^−/− value to the WT value. The data are mean ± standard deviation (SD) (n = 3 for each genotype). (C) CD43⁻ naive splenic B cells were isolated from WT, Aid^+/−, and Aid^−/− adult mice and stimulated with lipopolysaccharide (LPS) and interleukin 4 (IL-4). Total protein was extracted from stimulated cells and western blot against Aid and Vinculin were performed. (D) Whole splenic cells were collected from WT, Aid^+/−, and Aid^−/− adult mice around 6 months old. Total DNA was extracted from these cells and dot-blot assay was performed to detect 5hmC. Membrane was stained with MB to detect DNA loading control. Blot density was quantified by Image J software using blots derived from 100 ng of DNA. Right bar graph shows the ratio of the 5hmC value to the MB value. The data are mean ± SD (n = 3 for each genotype). MB, methylene blue; SPL, spleen.