Nupage lds

A range of 13–210 ng of kinase and the 200 ng of substrate were incubated at 30 °C for 30 min along with 2 mm DTT, 20 mm Tris, 1 mm EGTA, 1 mm MgCl₂ and 1 μl of 2 μCi/μl [γ-32P]. 4 × NuPAGELDS (Invitrogen, Thermo Fisher Scientific) sample buffer was added to the samples, then boiled at 95 °C for 5 min and resolved on a SDS–PAGE gel, which were coomassie stained. Signal was visualized by autoradiography. The proteins bands of interest were then excised and 32P incorporation was quantified by Cerenkov counting in a scintillation counter.

PMSA products were transferred from the reaction tubes to clean Eppendorf tubes and digested by adding proteinase K (PK) (Roche) at 25 µg/ml for 1 h at 42 °C in an oven (Nahita). Immediately after digestion, samples were centrifuged at 19,000g at 4 °C for 15 min, the supernatant was discarded and the pellet resuspended and washed with at least 700 µl of PBS (Fisher Bioreagents). After washing, samples were centrifuged for additional 5 min at 19,000g and 4 °C, supernatant discarded, and the pellet resuspended in 15 µl of loading buffer 4 ×  (NuPage LDS, Invitrogen), previously diluted to 1 × with PBS. PK-resistant PrP detection was done through electrophoresis and total protein staining. For that, PK-digested and concentrated samples in loading buffer were boiled for 10 min at 100 °C and loaded onto 4–12% acrylamide gels (NuPAGE Midi gel, Invitrogen Life Technologies), subjected to electrophoresis for 1 h and 20 min (10 min at 70 V, 10 min at 110 V and 1 h at 150 V) and stained with BlueSafe (NZYTech) for 1 h at room temperature. The same procedure was done for the determination of PK resistance of one of the PMSA products, but using 25, 100, 200, 500, 1000, 2000, 2500 and 3000 µg/ml of PK in a previously aliquoted sample. Additionally, PK digestions were also performed in one case using 25 µg/ml of PK at room temperature and 42 °C, for 6 and 24 h without shaking.

Four weeks after STZ injections, the mice were killed by decapitation without anaesthesia, as anaesthesia can lead to hypothermia-induced tau hyperphosphorylation65 (link). Brains were immediately removed and the tissues dissected on ice, frozen on dry ice, and kept at −80 °C until they were processed as described66 (link). Briefly, dissected brain structures (hippocampus and cortex) were homogenized, without thawing, in 5 times volume/weight of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340, Sigma-Aldrich, St. Louis, MO)), using a mechanical homogenizer (TH, Omni International, Marietta, GA). Samples were then centrifuged for 20 min at 20,000 g at 4 °C. The supernatant was recovered, diluted in sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich), boiled for 5 min. and kept at −20 °C.

Cells were lysed in lysis buffer and denatured in 1× NuPage LDS (Invitrogen) sample buffer supplemented with 25 mM DTT for 10 min at 70°C. The samples were separated on 4–12% Bis-Tris polyacrylamide gel and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). The blots were incubated with horseradish peroxidase-conjugated immunoglobulin’s (DAKO) and developed with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) before images were obtained with Odyssey Fc Imaging System (LI-COR). TLR8 overexpression in THP-1 subclones was confirmed by Western blotting using rabbit antihuman TLR8 XP mAb [Cell Signaling Technology (CST), no. D3Z6J]. Activation of IRF3 was done using rabbit antiphospho-IRF3 mAb (CST, no. D6O1M). Anti GAPDH (Abcam, #ab8245) served as a loading control.

Three-μl aliquots of α-synuclein PMCA or non-PMCA control samples were either mixed with 1 μl of 4× loading buffer (NuPAGE LDS®; Invitrogen) and incubated at 100 °C for 10 min (for SDS gels) or mixed with 1 μl of 4× native loading buffer (NativePAGE®; Invitrogen), and 3.5 μl of the mixture was loaded into 4–12% SDS (Bis-Tris)- or native gels (Invitrogen). Either low molecular mass standard (Bio-Rad) or SeeBlue Plus2 (Invitrogen) protein ladders were used as molecular mass markers for Bis-Tris gels, whereas NativeMark® unstained protein standards were used for native gels. In some cases gels were stained using only Coomassie Blue, whereas in other experiments proteins were transferred onto PVDF Immobilon membranes (Millipore), and α-synuclein was visualized by incubation with monoclonal or polyclonal anti-α-synuclein antibodies. Chemiluminescence was induced by ECL-Plus (Pierce) and recorded with the Alliance software (Uvitec Cambridge).

Extra virgin olive oil was from the Arbequina variety and had high oleic acid content (71.9% in weight). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), iron oxide magnetic nanoparticles (5 mg/mL in toluene), and IND were purchased from Sigma-Aldrich (St. Louis, MO, USA); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG-DSPE) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). DSC and PEG-DSPE were dissolved in chloroform at 20 mg/mL. Olive oil was diluted in chloroform at 100 mg/mL. Spectra-Por^® Float-A-Lyzer^® G2 was purchased from Spectrum Labs (Rancho Dominguez, CA, USA). The aqueous phase used to prepare the nanoemulsions was Hepes sodium salt buffer solution (10 mM, pH 7.2). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the sample buffer NuPAGE^® LDS (Invitrogen, Carlsbad, CA, USA) was used. Double distilled water was used in the preparation of all the solutions. Organic solvents were of analytical grade.