Figure 4.

The effects of PLPHP deficiency on protein-depleted and protein-enriched cell fractions.A, protein and PLP recovery after fractionation of HEK293 cells cultured with 19.4 μM PN for 96 h. B, B₆ vitamer distribution between low-molecular weight (filtrate, relatively protein-free) and high-molecular weight (concentrate, protein-rich) fractions in control and PLPHP-deficient fibroblasts. C, B₆ vitamer distribution between low- and high-molecular weight fractions in control and PLPHP-deficient HEK293 cells. D, Western blot showing PLPHP protein expression in control HEK293 cells cultured with no vitamin B₆ or 19.4 μM PN for 96 h. Data are means from n = 3 to 6 (A); n = 6 (PN) and n = 3 (no B₆) (B); n = 6 to 12 (PN) and n = 3 to 6 (no B₆) (C); and n = 2 (D) per cell line and condition, ±SD. ∗p < 0.05, ∗∗∗p < 0.001, ^#p < 0.05, and ^Xp < 0.05 compared to control 1, 2, and 3, respectively (B); ∗p < 0.05 and ∗∗p < 0.01 compared to control, and ^##p < 0.01 compared to PLPHP KO1 (C). All comparisons are at the same culturing condition (one way ANOVA with Tuckey’s post hoc test). PLP, pyridoxal 5′-phosphate; PLPHP, pyridoxal 5′-phosphate homeostasis protein; PN, pyridoxine.