Table 1.
Demographic and clinical features of cases (n = 28).
Fig 1.
Phospho-SP4 S770 is increased in peripheral blood mononuclear cells of patients with first-episode psychosis compared to control subjects and this increase is not present in those patients with lithium prescription.
Immunoreactivity of phospho-SP4 S770, SP4 and β-actin was determined in the same protein extracts from peripheral blood mononuclear cells (PBMC) of control (C, n = 14), and first-episode psychosis (FEP, n = 14) individuals. The resultant bands were quantified by densitometry. Both phospho-SP4 (pSP4) and SP4 were normalized to β-actin values and referred to a standard sample in two independent immunoblot analysis for pSP4 and SP4. pSP4/SP4 ratio was calculated for these samples by referring the mean normalized phospho-SP4 immunoreactivity levels to the mean normalized total SP4 immunoreactivity. Final pSP4/SP4 values were normalized to the mean of control group. Levels of total SP4 immunoreactivity normalized to β-actin were reported previously [19]. (A) Images show representative pSP4, SP4, and β-actin immunoblots from four control individuals and four FEP subjects. Patients FEP2 and FEP4 were both receiving lithium treatment. (B) The graph shows the mean and standard deviation of the normalized pSP4/SP4 ratio for control and FEP groups. Each value represents the mean of two independent analyses. One sample in FEP group had to be excluded from the analysis due to almost undetectable levels of pSP4 and SP4 (n = 13). Statistical analysis was performed using one-tailed Mann-Whitney test. (C) The graph shows the mean and standard deviation of normalized pSP4/SP4 ratio for FEP patients without lithium treatment (Untreated, n = 7), and FEP patients with lithium treatment (Treated, n = 6). Each value represents the mean of two independent analyses. An outlier was detected for pSP4/SP4 ratio in the lithium treated group using the Peirce criterion and therefore excluded from the analysis (n = 5). Statistical analysis was performed using two-tailed Mann-Whitney test. (D) The graph shows the mean and standard deviation of normalized total SP4 immunoreactivity for FEP patients without lithium treatment, and FEP patients with lithium treatment. Each value represents the mean of two independent analyses. Statistical analysis was performed using two-tailed Mann-Whitney test. (*p<0.05; n.s., not significant).
Table 2.
Influence of demographic and clinical characteristics on pSP4/SP4 ratio in peripheral blood mononuclear cells in first-episode psychosis and control subjects.
Fig 2.
SP4 phosphorylation at S770 is reduced by lithium chloride treatment.
A. Cerebellar granule neurons were treated with serum-free media containing 25mM KCl for two hours in the presence of the indicated concentrations of lithium chloride (A), valproic acid (VPA) (B) or olanzapine (OLZ) (C). 14 μg (A) or 5 μg (B and C) of a total protein extracts were analyzed by Western blot using antisera to phospho-SP4 S770, total SP4, phospho-GSK3β S9 (a positive control for lithium treatment) (A), phospho-Tau S396 (a positive control for VPA) (B), p42/44 MAPK (a positive control for OLZ) (C) and GAPDH as a loading control. The graphs show the densitometric quantification of S770 phosphorylated SP4 relative to the levels of total SP4. Statistical analysis was performed using ANOVA followed by Bonferroni’s post hoc comparison between untreated condition and the different doses of drugs. N = 3 (**p<0.01).