Alkaline phosphatase conjugated secondary antibody

PrP^Sc, purified as previously described [75 ], hamster ScBH at a dilution of 10⁻¹, α-synuclein, or Lewy bodies isolated from the brains of a Lewy body dementia patient, as described [76 (link)], were pretreated with saline or BrioHOCl solutions as indicated in the figure legends. Following treatment, samples were diluted with equal volumes of 2X sample buffer (125 mM Tris-HCl pH 6.8, 5% glycerol, 6 mM EDTA, 10% SDS, 0.04% bromophenol blue, 48% urea, 8% 2-mercaptoethanol) and boiled for 5 min. Equal volumes of samples were run on 10% Bis-Tris NuPAGE gels (Invitrogen) and used for subsequent Deep Purple protein stain per manufacturer’s instructions (GE Healthcare) or the protein transferred to an Immobilon P membrane (Millipore) using the iBlot Gel Transfer System (Life Technologies). PrP was detected using rabbit PrP antisera R30 (1:10,000; residues 90–104) [77 (link), 78 ] and alkaline-phosphatase conjugated secondary antibody (1:5000; Jackson ImmunoResearch). α-Synuclein was detected using mouse Anti-α-Synuclein Clone 42 antibody (1:1000; BD Transduction Laboratories) and alkaline-phosphatase conjugated secondary antibody (1:2000; Jackson ImmunoResearch). Tau was detected using a tau antibody (anti-tau ab64193, Abcam) as the primary antibody.

To obtain nuclear protein extracts from MSCs, 24 h after culture on substrates with distinct stiffness, cells were washed with PBS and then scraped in Laemmli buffer. The subcellular fractionation was done following the protocol described in^{75 (link)}, and nuclear fractions were collected into microtubes and then heated at 95 °C for 5 min. Next, the samples were spun down and passed ten times through a 25 G needle and further analyzed by Western blot. Proteins were separated by SDS-PAGE [12.5% (w/v) acrylamide–bisacrylamide (Bio-Rad) gels] and transferred onto PVDF membranes that were subsequently probed with specific antibodies against H4K16ac (Abcam) and H4 (Cell Signaling) followed by the incubation with the respective alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch). The membranes were incubated for 5 minutes with enhanced chemifluorescence substrates (ECF – GE Healthcare) and imaged in a Molecular Imager FX Pro Plus system (BioRad) using the Quantity One software (BioRad). The acquired images were analyzed with Image Lab software, version 5.1 (BioRad).