(A) Birth ratio of the obtained mice. A total of 316 mice were obtained by the mating shown in Supplemental Figure 1D, and the ratios of the 4 genotypes in male, female, and total mice are shown. Statistical analyses were performed with Pearson’s χ² test. Male: P = 0.55; female: P = 0.59; total: P = 0.90; ns: not significant (P > 0.05). (B) ITPA protein expression in P16 or P17 male Itpa-cKO (Itpa^fl/fl/Nes-Cre) and control (Itpa^fl/fl) mouse tissues. The ITPA protein levels in the tissue extracts from 3 Itpa-cKO and 3 control male mice were detected by Western blotting with anti-ITPA antiserum, quantified using cerebrum extract from 1 control mouse as a common standard to make a standard curve, and normalized by GAPDH levels. The ITPA expression levels in Itpa-cKO relative to those in control samples are shown for each tissue as the mean ± SD. Five micrograms of total protein of each sample was loaded in each lane. The original blot images are shown in Supplemental Figure 2. Statistical analyses were performed with Welch’s t test. P > 0.05 for each tissue. (C) Immunohistochemistry of P16 male mouse brains with anti-ITPA antibody. Images of sagittal (upper), coronal including cerebrum (middle), and coronal including cerebellum (lower) sections of paraffin-embedded brains of control male mice (left upper, Itpa^+/fl/ Nes-Cre; left middle, Itpa^+/fl; left lower, Itpa^+/fl/Nes-Cre) and Itpa-cKO male mice (right) are shown. Scale bar: 1 mm. (D) Inosine content in RNA from brain subregions and liver. The inosine content in the total RNA samples that did not contain any small RNA molecules extracted from the cerebral cortex, cerebellum, hippocampal formation, and liver of P16 control mice (Itpa^fl/fl) and Itpa-cKO mice are shown as the average inosine content per 10⁶ guanosine with standard deviations (n = 4). Statistical analyses were performed with Welch’s t test. Cerebral cortex: ****P < 0.0001; cerebellum: **P = 0.0019; hippocampal formation: ****P < 0.0001; liver: P = 0.30.