Detection of phascogale PrP^C with anti-PrP monoclonal antibodies. (A) IlFigurelustration of the prion protein and anti-PrP antibody epitope binding sites. SP = signal peptide; OR = octapeptide repeat region; HR = hydrophobic region; Gly = N-glycosylation sites; S-S; disulphide bridges. Antibodies: SAF32, SAF72, ICSM18. (B) Western blot analysis of 10% (w/v) P. calura brain homogenates treated with (+) or with no proteinase K (PK) using anti-PrP antibodies SAF32 and SAF70. (C) Western blot analysis of 10% (w/v) P. calura, mouse (M. musculus), hamster (Cricetinae), and human (H. sapiens) brain homogenates using anti-PrP antibodies SAF32 and SAF70. (D) Western blot analysis of 10% (w/v) P. calura brain homogenates derived from adult male breeders (AMB), adult male non-breeders (AMNB), male juvenile (MJ), a female juvenile (FJ), and an adult female (AF) using anti-PrP antibodies SAF32 and SAF70. Representative of 3 independent experiments.

Multiple PrP^C sequence alignment of phascogale and other marsupials. Species including phascogale (Phascogale calura), koala (Phascolarctos cinereus), Tasmanian devil (Sarcophilus harrisii), Tammar wallaby (Macropus eugenii), common wombat (Vombatus ursinus), possum (Trichosurus vulpecula), and opossum (Monodelphis domestica). Fully conserved between all species: * (asterisk); Strongly conserved between species: : (colon); Weakly conserved between species: . (period); Repeats: #1 (blue), #2 (red), #3 (green), #4 (purple), #5 (orange).

Interspecies phylogenetic relationship of marine, amphibian, reptilian, avian and mammalian species. The PrP^C instability index (II) is recorded next to each reported species. The colours in the phylogenetic tree represent different taxonomic groups of animals, helping to visually distinguish their evolutionary relationships: (i) Blue: Represents aquatic species, specifically fish and amphibians, such as the Japanese rice fish (Oryzias latipes), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis). (ii) Light-Green: Represents reptiles and birds, including species like the king cobra (Ophiophagus hannah), saltwater crocodile (Crocodylus porosus), and wild turkey (Meleagris gallopavo). (iii) Yellow: Represents monotremes and marsupials, such as the platypus (Ornithorhynchus anatinus), common wombat (Vombatus ursinus), and Tasmanian devil (Sarcophilus harrisii). (iv) Green: Represents placental mammals, including humans (Homo sapiens), elephants (Loxodonta africana), and mice (Mus musculus). (v) Orange: Represents various mammalian species with higher protein instability index (II), such as dogs (Canis lupus familiaris), rhinos (Diceros bicornis), and whales (Balaenoptera acutorostrata scammoni).

Multiple PrP^C sequence alignment of phascogale with mammalian and avian species. Species included phascogale (Phascogale calura), human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), hamster (Mesocricetus auratus), bank vole (Myodes glareolus), rabbit (Oryctolagus cuniculus), sheep (Ovis aries), goat (Capra hircus), bovine (Bos taurus), deer (Cervidae), elk (Cervus), cat (Felis catus), dog (Canis lupus familiaris), camel (Camelus dromedarius), and chicken (Gallus gallus domesticus). Fully conserved between all species: * (asterisk); Strongly conserved between species: : (colon); Weakly conserved between species: . (period); Repeats: #1 (blue), #2 (red), #3 (green), #4 (purple), #5 (orange), #6 (pink).

Multiple PrP^C sequence alignment of phascogale with mammalian and avian species. Species included phascogale (Phascogale calura), human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), hamster (Mesocricetus auratus), bank vole (Myodes glareolus), rabbit (Oryctolagus cuniculus), sheep (Ovis aries), goat (Capra hircus), bovine (Bos taurus), deer (Cervidae), elk (Cervus), cat (Felis catus), dog (Canis lupus familiaris), camel (Camelus dromedarius), and chicken (Gallus gallus domesticus). Fully conserved between all species: * (asterisk); Strongly conserved between species: : (colon); Weakly conserved between species: . (period); Repeats: #1 (blue), #2 (red), #3 (green), #4 (purple), #5 (orange), #6 (pink).