Detection of PrP^res in fCJD^V180I and VPSPr with 1E4 and 3F4.

<p>(<i>A</i>–<i>D</i>) Brain homogenates from six fCJD^V180I cases (four 129MM and two 129MV) were treated with different amounts of PK from 0 to 100 µg/ml prior to Western blotting with 3F4 (<i>A</i> and <i>C</i>) or 1E4 (<i>B</i> and <i>D</i>). (<i>E</i>) Comparison of PrP^res from fCJD^V180I, VPSPr, and sCJD probing with 1E4.</p

Detection of two individual monoglycosylated PrP either at N181 or N197.

<p>(<i>A</i>) and (<i>B</i>) sCJDMM2 (lanes 1, 2), two VPSPr (129MM: lanes 3, 4; 129MV: lanes 5, 6), fCJD^T183A (lanes 7, 8) and fCJD^V180I (lanes 9, 10) treated with PK or PK plus PNGase F were probed with Bar209 (<i>A</i>) or V14 (<i>B</i>). (<i>C</i>) PrP from VPSPr, fCJD^V180I, and sCJD was examined with V14 (lanes 1–3) or Bar209 (lanes 4–6). (<i>D</i>) Ratio of mono- (either mono181 or mono197) to unglycosylated PrP by densitometric analysis based on three independent experiments, one of which is presented in (<i>C</i>). The black bar represents mono181:unglyc PrP, while the grey bar represents mono197:unglyc PrP from sCJD, fCJD^V180I or VPSPr. *** <i>p</i> <0.005.</p

Comparison of glycosylation, proteolysis and distribution of PrP^T183A, PrP^V180I, or PrP^Wt expressed in cultured cells.

<p>Cell lysates from cultured M17 cells expressing PrP^T183A, PrP^V180I, or PrP^Wt were treated with or without PK and/or PNGase F prior to Western blotting with 3F4 (<i>A</i>) or 1E4 (<i>B</i>). Subcellular localization of PrP^T183A, PrP^V180I and PrP^Wt (<i>C</i>, <i>D</i>, and <i>E</i>). Immunofluorescence staining of cells using 3F4 for PrP (green) and calnexin for ER (red). Virtually all PrP^T183A was colocalized with calnexin (<i>C</i>). PrP^V180I was also colocalized with calnexin, but was found on cell surface equally (<i>D</i>). PrP staining was mostly found on the cell surface in cells expressing PrP^Wt (<i>E</i>). Scale bars: 25 µM.</p

Schematic diagram of glycoform-selective prion formation pathway of PrP^V180I in the brain.

<p>Four different glycoforms from the PrP^V180I mutant allele (gray) are converted into PK-resistant PrP^res in cells. However, in the presence of four glycoforms from the wild-type allele (black) in the brain, mono181 and diglycosylated PrP^Wt bind to their mutant counterparts, respectively and the PrP^Wt-PrP^V180I complexes are removed from the prion conversion pathway due to possible dominant-negative inhibition. So, only mono197 and unglycosylated PrP are converted into PrP^res, whereas mono181 and diglycosylated PrP are not. With Western blotting (WB), 1E4 is able to detect three PK-resistant PrP^res including the diglycosylated form, while 3F4 does not in cells. In contrast, in the brain tissues, 3F4 detects mono197 and unglycosylated PrP^res but not diglycosylated and mono181 PrP^res. 1E4 detects five PK-resistant PrP^res without diglycosylated form.</p

Detection of PrP^Sc before and after PK-treatment.

<p>PrP in P2 fractions from sCJD, VPSPr and fCJD^V180I was subjected to g5p-capture and treatment with or without PK prior to Western blotting with Bar209 (<i>A</i> and <i>B</i>) or V14 (<i>C</i> and <i>D</i>).</p