Anti-LC3 mAb

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	The LC3-II / LC3-I ratio was 10- to 40-fold lowerr in rapamycin-non treated cells. Dilute primary at 1:1000

Protocol tips
The LC3-II / LC3-I ratio was 10- to 40-fold lowerr in rapamycin-non treated cells. Dilute primary at 1:1000

Publication protocol

The drug treated cells were harvested with lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate and 2 mM EDTA). After 2 min of centrifugation at 10,000 × g, the supernatant was collected and its total protein concentration was measured using the BCA protein assay kit (Nacalai Tesque). To detect PrPSc, the protein concentration was adjusted to 5 mg/ml and the samples were digested with 20 μg proteinase K (PK; Sigma)/ml at 37°C for 30 min, followed by boiling for 10 min with sample buffer (50 mM Tris-HCl, pH 6.8, containing 5% glycerol, 1.6% SDS, 100 mM dithiothreitol and a moderate amount of bromophenol blue). After SDS-polyacrylamide (15%) gel electrophoresis, the proteins were transferred onto a PVDF membrane (Immobilon-P; Merck Millipore) which was blocked with 5% skim milk in TBST (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. The membrane was then reacted with diluted primary (1:1000) and HRP-conjugated secondary antibodies (1:5000). Immunoreactive bands were visualized by ECL prime (GE Healthcare). To quantify the signals, the intensity of each band was measured using the NIH image J software. A detailed description of the methods was previously provided [22].

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