Amersham hybond p 0.45 pvdf membranes

Cell lysis, PK profiling and immunoblot was performed as described previously^{57 (link)}. Briefly, confluent cells were lysed in chilled cell lysis buffer (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; 10 mM EDTA; 0.5% Triton X-100; 0.5% sodium deoxycholate (DOC)) for 10 minutes. For PK digestion, 50% of the cell lysate was incubated with 20 μg/ml PK (Roche, Germany) for 30 min at 37 °C. PK digestion was stopped with 0.5 mM pefabloc protease inhibitor (VWR), and the samples were precipitated with 5 volumes of methanol. For non-PK digested samples (−PK), pefabloc was added directly and samples precipitated with methanol. Precipitated proteins were re-suspended in appropriate volume of TNE buffer (50 mM Tris-HCl pH 7.5; 5 mM EDTA; 150 mM NaCl). Samples were run on 12.5% SDS-PAGE, and electro-blotted on Amersham Hybond P 0.45 PVDF membranes (Amersham, USA). The blots were developed using Luminata Western Chemiluminescent HRP Substrates (Millipore, USA). PrP^c and PrP^Sc was detected with anti-PrP monoclonal antibody (mAb) 4H11 as described previously^{58 (link)}. Peroxidase-conjugated goat anti-mouse HRP secondary antibodies were from Jackson ImmunoResearch, USA. Anti-β-actin (Sigma Aldrich, USA) was used for loading control.

Immunoblot analysis was done as previously described^{40 (link)}. Briefly, confluent cells were lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; 10 mM EDTA; 0.5% Triton X-100; 0.5% sodium deoxycholate (DOC)) for 10 min. Aliquots of lysates were incubated with PK (20 µg/ml) for 30 min at 37 °C. PK was stopped by addition of proteinase inhibitors (0.5 mM Pefabloc) and directly precipitated with methanol. For samples without PK treatment, proteinase inhibitors were added directly and precipitated with methanol. Precipitated proteins were re-suspended in TNE buffer (50 mM Tris-HCl pH 7.5; 150 mM NaCl; 5 mM EDTA). Samples were run on 12.5% SDS-PAGE, electro-blotted on Amersham Hybond P 0.45 PVDF membranes (Amersham, USA) and analyzed in immunoblot, using Luminata Western Chemiluminescent HRP Substrates (Millipore, USA).

Methanol precipitated cell lysates were centrifuged, and pellets resuspended in TNE buffer (50 mM Tris-HCl pH 7.5; 150 mM NaCl; 5 mM EDTA). 12.5% SDS-PAGE was used. Gels were electro-blotted on Amersham Hybond P 0.45 PVDF membranes (Amersham, 10600023) and incubated with the desired antibodies. Luminata Western Chemiluminescent HRP Substrates from Millipore (WBLUF0100) was used for development. Densitometry was done using ImageJ program (NIH, USA). LC3-II signals were normalized to actin for quantification.