Sds page novex gels

To analyze iNOS expression, cells in each well were lysed in 200 μL lysing buffer MPER (Thermo Scientific, cat #78501) supplemented with protease inhibitor cocktail (Roche, cat #04693159001), then homogenized by brief sonication for 30 seconds (Misonix S-4000 microplate horn, Qsonica), supplemented with 4X SDS buffer (200 mM Tris-HCL, pH 6.8, 8% SDS, 40% Glycerol, 4% beta mercaptoethanol, 50 mM EDTA, 0.08% bromophenol blue) and incubated in boiling water for 10 minutes. Samples were analyzed using precast SDS-PAGE NOVEX gels (Thermo Fisher, cat #NP0343BOX), immunoblotted as described elsewhere and stained using anti-iNOS antibody (BD Biosciences, cat #610328) at 1:3000 dilution overnight. Western blots were developed using chemiluminiscence substrate (Thermo Scientific, cat # 1859674 & 1859675) and visualized using a FluorChem M system (ProteinSimple). The signal intensities were digitized using AlphaView software (ProteinSimple). Blots probed with anti-β actin antibody (Sigma cat #A5441) served as protein loading controls. Data from three independent experiments were used for densitometry analysis of changes in iNOS expression.

In preliminary experiment, a time dependent degradation of Iκβα was established. Briefly, BV2 cells were cultured in 24-well plates to 70–80% confluency, then treated with brain-derived 22L PrP^Sc, ds22L PrP^Sc or mock-purified materials for 10, 20, 30, 45 or 60 minutes. Cells treated with DPBS (Cat #INV-14190144, Life Technologies) or LPS (100 ng/ml, E. coli 0111:B4) served as negative and positive controls, respectively. Cells were harvested in 200 µl using the cell lysis buffer MPER (Cat #78501, Thermo Scientific), then supplemented with 4X SDS buffer, incubated in boiling water for 10 minutes and analyzed using precast SDS-PAGE NOVEX gels (Thermo Fisher, cat #NP0343BOX). Blots were stained using anti-Iκβα antibody (cat #9242S, Cell Signaling Technology) at 1:1000 dilution. Blots were re-probed with anti-β actin antibody, which served as protein loading control, using reblotting buffer (Millipore cat #2502). For analysis of Iκβα degradation at the fixed time point, BV2 cells cultured in 24-well plates were treated with brain-derived 22L PrP^Sc, ds-22L PrP^Sc, mock-purified materials, DPBS or LPS for 20 minutes. And analyzed as described above. β actin served as loading controls.