Disease-associated mutations in the prion protein impair laminin-induced process outgrowth and survival

 

Western blotting

Cell      extracts      were      prepared      by

homogenizing the cell pellet in lysis buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1

mM EDTA, 1% Triton X-100, 0.1% SDS,

and 1% sodium deoxycholate) containing a protease inhibitor mixture (complete protease inhibitor tablets; Roche Diagnostics). Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Millipore), and incubated with 1µg/mL 3F4 antibody followed by HRP- anti-mouse IgG. Immunoreactivity was revealed by enhanced chemiluminescence (ECL Plus; GE Healthcare).

Proteinase K resistance

Transfected CF10 and control cells were washed twice with cold PBS, scraped from

plate, pelleted by centrifugation, and lysed in cold buffer (0.5% Triton X-100, 0.5% sodium deoxycholate in PBS, 10 mM Tris-

HCl, 10 mM NaCl and 10 mM EDTA). For proteolysis    experiments,    lysates    were

shared (25% for control and 75% for digestion) and incubated for 30 min at 4°C with  proteinase  K  (PK)  (Roche)  at  the

concentrations indicated. Samples were precipitated with methanol. One volume of

sample was mixed with 2.5 volumes of methanol. After 2 hr at -80 ºC, the mix was

centrifuged at 1000 xg for 30 min at 4ºC, the supernatant was discarded, and pellets were  air  dried.  The  dried  pellets  were

dissolved in resuspension buffer (150 mM NaCl, 50 mM Tris-HCl, 50 mM EDTA).

The presence of residual, proteinase K- resistant PrPC was evaluated by…