4000r imaging station

Western blot detection of PrP^Sc from brain homogenate was performed as described previously [76 ]. Briefly, brain homogenate (5% w/v) in PMCA conversion buffer is digested with proteinase K (PK) at a final concentration of 100, 200, or 400 μg/ml (Roche Diagnostics Corporation, Indianapolis, IN) at 37°C for 1 or 24 hours. The samples were either enriched for PrP^Sc as described previously [77 (link)] or an equal amount of sample buffer containing 4% (v/v) 2-mercapto ethanol and 8% (w/v) SDS was added and the mixture was incubated at 100°C for 10 minutes. Prion protein was detected with the anti-PrP antibody 3F4 (final concentration of 0.1 μg/ml; Chemicon; Billerica, MA) and HRP-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch; West Grove, PA). The Western blot was developed with Pierce Supersignal West Femto Maximum Sensitivity Substrate according to manufacturer instructions (Pierce, Rockford, IL), imaged on a Kodak 4000R Imaging Station (Kodak, Rochester, NY) and analyzed using Kodak Molecular Imaging Software v.5.0.1.27 (New Haven, CT). Statistical analysis was performed using Prism 6.0 for Mac (GraphPad Software Inc., La Jolla, CA).

Western blot analysis was performed as previously described [76 (link)]. Briefly, samples were digested with 100 U/ml of proteinase K (PK) at 37°C for 30 minutes with constant agitation (Roche Diagnostics Corporation, Indianapolis, IN). The PK digestion was terminated by incubating the samples at 100°C for 10 minutes in gel loading buffer (4% w/v SDS, 2% v/v β- mercapto ethanol, 40% v/v glycerol, 0.004% w/v Bromophenol blue, and 0.5 M Tris buffer pH 6.8). Following size fractionation on 4–12% Bis-Tris gel, the proteins were transferred to immobilon P and Western blot analysis were performed using the anti-PrP antibody 3F4 (final concentration of 0.1 μg/ml; Chemicon; Billerica, MA) to recognize hamster prion protein. The Western blot was developed with Pierce supersignal west femto maximum sensitivity substrate according to manufacturer’s instructions (Pierce, Rockford, IL) and imaged on a Kodak 4000R imaging station (Kodak, Rochester, NY). The abundance and migration of PK resistant PrP^Sc was determined using the Kodak molecular imaging software v.5.0.1.27 (New Haven, CT). Cropped images are indicated by a vertical line and are from the same exposure of the same blot unless otherwise noted. The signal intensity of the unglycosylated PrP^Sc polypeptide as a function of migration distance was determined using the Kodak molecular imaging software v.5.0.1.27 (New Haven, CT).

Western blot analysis was performed as our previous report16 (link). Equal amounts of protein measured by BCA protein assay kit were mixed with 6 × SDS reducing sample buffer and boiled for 5 minutes before loading. The proteins (50 μg/lane) were separated on an SDS polyacrylamide gel and transferred electronically to PVDF membranes. After blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 h, the membranes were incubated with primary antibodies against COX2 (1:200), SOD1 (1:200), PDRX4 (1:200) or β-actin (1:1000) at 4 °C overnight, then incubated with HRP conjugated goat anti-mouse or goat anti-rabbit IgG (1:1000) for 1 h. The signals of the bands were detected with a chemiluminescence reagent (ECL, GE Healthcare) using Kodak 4000R imaging station (Eastman Kodak, Rochester, NY).