Semi dry electrotransfer system

PMCA samples were run on Criterion XT 12% Bis-Tris precast gels (Biorad, Hercules, CA, USA), electrotransferred onto nitrocellulose membranes with the semi-dry electrotransfer system (Biorad) and probed with biotinylated Sha31 anti-PrP monoclonal antibody62 (link), as described above. PrP^C content of the cell lysates was determined by western blotting with SAF3462 (link) anti octarepeat region of PrP. Quantification was determined with the GeneTools software after acquisition of the signals with a GeneGnome digital imager.

Protein was extracted from cell lines using RIPA buffer (Sigma) supplemented with a protease inhibitor cocktail (cOmplete Mini EDTA-free, Roche) and a phosphatase inhibitor cocktail (PhosSTOP EASYpack, Roche). A standard western blot protocol was utilized with a miniProtean electrophoretic system (BioRad) and a semi-dry electrotransfer system (BioRad). Primary antibodies against FGFR4 (#8562, Cell Signaling), AKT (#9272, Cell Signaling), pAKT (#9271, Cell Signaling), ERK1/2 (#9102, Cell Signaling), pERK1/2 (#9101, Cell Signaling), STAT3 (#9139, Cell Signaling), pSTAT3 (#9145, Cell Signaling), N-cadherin (#3195, Cell Signaling), Vimentin (#5741, Cell Signaling), Twist (#46702 S, Cell Signaling), Snail (#3879 S, Cell Signaling) and α-tubulin (#T9026, Sigma) were used. α-tubulin protein expression was used as the loading control. Horseradish peroxidase-conjugated secondary antibodies were used for chemiluminescence-based detection of protein expression in the ChemiDoc detection system (BioRad). No grouping of gels/blots cropped from different parts of the same gel, or from different gels, fields, or exposures was performed.

Protein samples were thawed to room temperature and amount of total protein quantified using a BCA protein assay (ThermoFisher). Fifty microgram of total protein was separated by SDS-PAGE (Bio-Rad, CA, United States) and transferred to polyvinylidene difluoride membranes (Bio-Rad) using a semi-dry electrotransfer system (Bio-Rad). The chemiluminescence (ECL) HRP substrate membranes were then blocked in 3% bovine serum albumin (BSA; Sigma) in 0.1% Tween-20 in TBS (TBS-T) for 1 h at room temperature. The blot was then cut into separate pieces based upon the size distribution and the appropriate gel section blotted overnight at 4°C in 3% BSA in TBS-T with the following antibodies: beta-actin (Cell Signaling Technology, MA, United States; Cat #13E5; 1:1,000), pS6 Ser 235/236 (Cell Signaling Technology; Cat #2211; 1:1,000) and cleaved caspase 3 (CC3; Cell Signaling Technology; Cat # 5A1E; 1:200). The following day the blots were washed 3 × 5 min in TBS-T and subsequently blotted with anti-rabbit secondary (Jackson Immuno Research Laboratories, PA, United States; 1:5,000) in 3% BSA in TBS-T for 1 h at room temperature. The blots were washed 3 × 5 min in TBS-T and incubated with SuperSignal West Dura chemiluminescence substrate (ThermoFisher), imaged on ChemiDoc XRS+ imager (Bio-Rad) and analyzed using the Quantity One software package (Bio-Rad).