Fig. 8. Excessive phosphorylation in L-MPZ mice.

a Phos-tag-western blotting of sciatic nerve homogenates (WT, 5 µg; Het and Hom 1 µg per lane) using anti-L-MPZ and anti-phospho-(Ser) PKC (PKC substrate) antibodies. Multiple phosphorylated-L-MPZ bands were found in both 10-week-old L-MPZ homozygous (Hom) and heterozygous (Het) mice compared with wild-type (WT) mice, in which only faintly stained bands were found. Anti-phospho-(Ser) PKC antibody recognized two L-MPZ-positive major bands (i, ii), suggesting that either a single (i) or two PKC-phosphorylation sites (ii) in L-MPZ were phosphorylated by PKC in sciatic nerves of L-MPZ Hom and Het mice. Higher smeared bands appear to be multiple-phosphorylated L-MPZ at other phosphorylation sites in addition to PKC phosphorylation. Three individual mouse samples in each group were used for anti-L-MPZ. The three lanes of anti-PKC substrate indicate representative samples in each group. b Each band intensity in a (anti-L-MPZ antibody) was measured and the ratio of phosphorylated L-MPZ bands (i, ii) to non-phosphorylated L-MPZ (bottom bands) were compared. L-MPZ mice showed an increased ratio of phosphorylated L-MPZ levels of these two bands especially in the sciatic nerve of Hom mice. c The ratio in amount of two phosphorylated L-MPZ bands (i, ii) per total L-MPZ-positive protein content was calculated and compared in different genotypes. Phosphorylated L-MPZ levels were increased in L-MPZ mice. *p < 0.05; **p < 0.01; ***p < 0.001 by ANOVA post-hoc with Tukey’s test. Data are presented as mean ± SE of three experiments. N (mice) in WT = 3, Het = 3, Hom = 3 (b, c).