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. 2024 Jun 10;13:RP93094. doi: 10.7554/eLife.93094

Figure 1. Role of pyridoxal 5’-phosphate phosphatase (PDXP) in hippocampal pyridoxal 5’-phosphate (PLP) homeostasis.

Figure 1.

(a) Age-dependent expression of pyridoxal kinase (PDXK) and PDXP in murine hippocampi. Left panels, representative western blots of three hippocampi for each genotype. The same blots were reprobed with α-actin antibodies as a loading control. The age of the investigated mice is indicated above the blots. Right panel, densitometric quantification of hippocampal PDXP and PDXK western blot signals, corrected by the corresponding actin signals. Young mice were 18–42 days of age, older mice were 252–351 days of age; n=7 individual hippocampi were analyzed per group. Data are mean values ± SD. Statistical analysis was performed with unpaired, two-sided t-tests; p-values are indicated. (b) Age-dependent, total PLP concentrations in isolated hippocampi of PDXP-WT and knockout of PDXP (PDXP-KO) mice. PLP was derivatized with semicarbazide and analyzed by HPLC. Each symbol represents the result of the PLP determination in an individual hippocampus. Data were fitted by Gaussian least-squares analyses. (c) Determination of protein-bound and protein-depleted PLP in PDXP-WT and PDXP-KO hippocampal lysates of young (18–42 days of age) and older mice (252–352 days of age). The number of analyzed hippocampi is indicated in the bars. Data are mean values ± SD. Statistical analysis was performed with two-way ANOVA and Tukey’s multiple comparisons test. Significant differences (adjusted p-values) in protein-depleted PLP levels are indicated. The exact age of analyzed mice is listed in Figure 1—source data 3. Source data are available for this figure.

Figure 1—source data 1. Western blot quantification (to Figure 1a).
Densitometric quantification of pyridoxal 5’-phosphate phosphatase (PDXP), pyridoxal kinase (PDXK), and actin levels in hippocampi of PDXP-WT mice, and of PDXK and actin levels in hippocampi of knockout of PDXP (PDXP-KO) mice.
Figure 1—source data 2. Quantification of pyridoxal 5’-phosphate (PLP) levels in hippocampi of pyridoxal 5’-phosphate phosphatase (PDXP)-WT and knockout of PDXP (PDXP-KO) mice (to Figure 1b and c).
High-performance liquid chromatography (HPLC)-based measurements of total PLP concentrations in isolated hippocampi of PDXP-WT and PDXP-KO mice, and of protein-bound and protein-depleted PLP levels in PDXP-WT and PDXP-KO hippocampal lysates.
Figure 1—source data 3. Analysis of total hippocampal pyridoxal 5’-phosphate (PLP) levels in pyridoxal 5’-phosphate phosphatase (PDXP)-WT and knockout of PDXP (PDXP-KO) mice.
Each value represents the result of the PLP determination in an individual hippocampus. Analysis for statistically significant differences between PLP levels in PDXP-WT and PDXP-KO hippocampi (all ages combined; two-tailed, unpaired t-test) p<0.0001. Bold table entries indicate those hippocampal extracts that were further separated for an analysis of protein-depleted and protein-bound PLP (see Figure 1c). Source data are available for this table.