664 Fig. 2 Western blot proﬁles of PrPSc (3F4 antibody) from different brain regions of a one sCJD MM 1?2C subject showing the type 2 protein only focally in the cerebral cortex (arrowheads) and b one sCJD MM 2C?1 subject showing a widespread PrPSc type 2 accumulation, a focal deposition of the type 1 protein (arrowheads) in the cerebral cortex, putamen, and thalamus, and a dominant type 1 in the cerebellum. Areas shown include the middle frontal gyrus (MFG), middle temporal gyrus (MTG), parietal cortex (PC), lateral occipital cortex (OCC2), insular cortex (INS), putamen nucleus (PUT), medial thalamic nuclei (TH1), and cerebellum (CE). Approximate molecular masses are in kilodaltons Fig. 3 Prevalence and regional distribution of PrPSc type 2 in a series of 57 MM 1?2C subjects. PrPSc type 1 was detected in all areas. Subjects are divided in 3 groups based on the number of areas with a detectable PrPSc type 2 signal on WB. For each group, the % of type 2 positive samples in each brain region is shown. The mean total type 2 ‘‘load’’ for each group, as deﬁned in ‘‘Materials and methods’’, is given in brackets. FC frontal cortex, TC temporal cortex, PC parietal cortex, OCC occipital cortex, Limbic CTX limbic cortices, Hipp ? AMG hippocampus?amygdala, STR striatum, HYP hypothalamus, TH thalamus, BRST brainstem, CE cerebellum MV subjects PrPSc types 1 and 2 concurrence in MV cases was detected in two phenotypically distinct groups of subjects. The 123 Acta Neuropathol (2009) 118:659–671 absence or presence of kuru plaques best distinguished the two groups. In subjects lacking kuru plaques, the con- currence of PrPSc types reproduced the features seen in MM 1?2C subjects. At variance with the MM group, however, the relative proportion of MV 1?2C and MV 2C?1 was signiﬁcantly different. Indeed, only one case had type 1 in all brain regions and the concurrent type 2 in 5, two had 12 and 14 areas with a mixed molecular phenotype, respectively (but a predominant type 1 load), while the remaining 3 showed a predominant type 2 accumulation (Table 3). PrPSc types 1 and 2 concurrence was more difﬁcult to assess in MV subjects with kuru plaques (i.e. MV 2K subtype) because of the presence of the 20 kDa band (Fig. 1), which we detected in virtually all samples from subcortical structures and in 75% of those taken from the neocortex (data not shown). Thus, a relatively weak 21 kDa signal cannot be considered proof of bona-ﬁde PrPSc type 1 concurrence, since it would also be compatible with a partially cleaved fragment of the 20 kDa PrPSc core [25]. As a consequence, we considered the 21 kDa band a bona-ﬁde type 1 only when its intensity was similar or higher than that of the 20 kDa fragment. Using these stringent criteria, we detected an undisputable type 1 signal in a least two cortical samples in only 2 of the 24 MV 2K cases analyzed (data not shown). Interestingly, at variance with the other MV 2K cases, the PrPSc proﬁle in these two cases included the so called 13 kDa CTF (C-terminal truncated fragment) (data not shown), which at variance with MV 2K is consistently found in both MM1 and VV1 cases [26].