Sensitivity of the CDNF-ELISA to human recombinant CDNF (Icosagen, Tartu, Estonia) was determined by calculating the mean response of ten blank samples and evaluating the mean plus three standard deviations on the standard curve. Specificity of the ELISA was tested by measuring cross-reactivity to recombinant mouse CDNF (R&D Systems) and to human recombinant MANF (Icosagen). GDNF-ELISA

The amount of GDNF protein in the brain samples was analyzed using the GDNF Emax® ImmunoAssay System (Promega, Madison, WI) according to the instructions from the supplier. Inhibitors,research,lifescience,medical The concentration of GDNF was compared with the total amount of protein in the sample. Statistical analysis All results are given as mean values with error bars showing the standard error of the mean (SEM). Statistical Inhibitors,research,lifescience,medical analyses were performed using PASW Statistics 18 (SPSS, Inc., Chicago, IL). For normally distributed data, differences between treatment groups were determined with one-way analysis of variance (ANOVA) followed by Tukey honestly significant difference (HSD) Inhibitors,research,lifescience,medical post hoc test. In cases when

Levene’s test for homogenicity gave statistical significance, Games–Howell post hoc test was selleck chemical Enzastaurin applied. Differences between treatment groups were selleck chemicals Romidepsin considered statistically significant if P < 0.05. Results Characterization of viral vector-mediated expression in intact animals Sensitivity and specificity of the in-house-built CDNF-ELISA Sensitivity of the CDNF-ELISA was determined to a minimum concentration of 10 pg/mL of recombinant human CDNF. In cross-reactivity tests, the assay recognized only 5% of mouse CDNF at the concentration Inhibitors,research,lifescience,medical 2 ng/mL. Human recombinant MANF gave no signal in the assay (highest tested concentration was 500 ng/mL). The ELISA readings of hCDNF in all the control rat brain samples (intact, AAV2-GFP- Inhibitors,research,lifescience,medical or vehicle-treated hemispheres) were under the detection limit of the assay. Time- and titer-dependent protein expression following AAV-CDNF injection in vivo The CDNF expression

following intrastriatal AAV2-CDNF injection was monitored with CDNF-ELISA and showed both time and titer dependence (Fig. 2). A five-time increase in the injected virus vector titer (from 2 × 108 to 1 × 109 vg) resulted in a statistically significant increase from about 160 pg of CDNF/mg of total protein to about 530 pg of CDNF/mg of total protein (P < 0.05, one-way ANOVA [F2,9 = 16.792, Dacomitinib P = 0.001] and Games–Howell post hoc test) (Fig. 2A). In the striatum (Fig. 2B), expression of hCDNF at about 180 pg/mg of total protein could be detected already 1-week postinjection (AAV2-CDNF 109 vg), followed by a quite robust increase in the expression at 2-week postinjection (approximately 490 pg of CDNF/mg of total protein). The expression remained stable until the end of the study (12-week postinjection), when the total amount of hCDNF in the dissected striatum was 1.3 ± 0.