Fig 2: Immunostaining patterns of BAMBI in the central nervous system of scrapie-infected Tg338 mice. Figure shows representative images of BAMBI immunostaining in the thalamus, hypothalamus, deep cerebellar nucleus and medulla oblongata (20 µm). Deposition type of BAMBI consisted of coarse granules found in the nuclei of cells, which were more evident in neurons of the thalamus, deep cerebellar nuclei of cerebellum (arrowhead and detail) and medulla oblongata. In addition, a diffuse neuropil staining was observed, remarkably strong in the hypothalamus but also present in the other brain regions studied.

Fig 3: Western blot quantification of BAMBI and CHGA proteins in the CNS of scrapie-infected sheep. (A) Membranes displaying the specificity of antibodies against BAMBI and CHGA proteins in the ovine thalamus (T), medulla oblongata (Mo), frontal cortex (Fc) and cerebellum (Cbl) of control and scrapie-infected sheep detected using Western blot. Each protein was cropped and grouped from different parts of the same gel or from different gels. Distinctive bands of ~30 kDa and ~50 kDa confirmed the specificity of the antibodies used against BAMBI and CHGA, respectively. (B) Western blot quantification of BAMBI in T and Mo and CHGA in Fc, Cbl and Mo. Density of immunoreactive bands was normalized for ß-Actin density band and is reported as arbitrary units. Data are expressed as means ± standard deviation. Quantification of density of bands did not reveal significant changes in any of the analysed areas, although CHGA displayed a trend to upregulation in scrapie animals (black bars) in Fc (p = 0.097). Differences between groups were determined using the Student´s t-test (? p < 0.1).

Fig 4: Semi-quantitative scoring of BAMBI in the central nervous system of scrapie-infected Tg338 mice. Figure includes a global graph (A) and comparative graphs (B) between clinical group (black bars) with preclinical (grey bars) and control group (grey-striped bars). Comparative graphs between the preclinical group and their control group, and between control groups, are not shown because no significant differences were detected. Score values (from 0: negative, to 5: staining present at its maximum intensity) evaluated in the frontal cortex (Fc), septal area (Sa), thalamic cortex (Tc), hippocampus (Hc), thalamus (T), hypothalamus (Ht), mesencephalon (Mes), cerebellum (which includes molecular layer (Ml), Purkinje layer (Pl), granular layer (Gl), white matter (Wm) and deep cerebellar nuclei (DCN)) and medulla oblongata (Mo). The differences between the experimental groups were determined using the Mann Whitney U test (* p < 0.05). Significant differences after Bonferroni correction for multiple tests are shown as # p < 0.05.

Fig 5: ELISA quantification of BAMBI and CHGA proteins in CSF of scrapie-infected sheep and BAMBI in CSF of CJD patients. (A) Intensity of BAMBI and CHGA signals in CSF of control (NC, round dots) and scrapie sheep at preclinical (PS, triangular dots) and clinical stage (CS, square dots). Protein BAMBI displayed an upregulation in CSF of scrapie-affected sheep at clinical stage (2465 ± 174 pg/mL) compared to scrapie sheep at preclinical stage (2103 ± 132 pg/mL, p < 0.01) and to control sheep (2114 ± 84 pg/mL, p < 0.01). No significant differences were detected between groups for CHGA protein. (B) Concentrations of BAMBI in CSF of control (ND, round dots) and CJD cases (square dots). This protein displayed increased concentrations in CJD (1771 ± 588 pg/mL) compared to ND (1370 ± 474 pg/mL, p < 0.01). N.S.: no statistically significant value. Differences between groups were determined using the Student´s t-test (** p < 0.01).