Xcell surelock system

Electrophoresis under denaturating conditions was performed using 8 cm 4–12 % Bis-Tris precast gels, using MES running buffer and Novex Sharp Standard, in an XCell Sure Lock system from Invitrogen (Carlsbad) according to manufacturer’s instructions. Western blot detection was performed using guinea pig serum against mumps virus which was cultured in Vero cells and purified by ultracentrifugation. ECL Prime Western Blotting Detection Reagent was used for detection, according to the manufacturer’s instructions (GE Healthcare). Detection of protein bands was performed using acidic Coomassie Brilliant Blue R250 solution. Protein bands were excised from the gel, trypsinized and peptides isolated and purified for MS analysis as described [64 (link)]. Experiments were performed with at least two sample replicates.

Protein samples in reducing sample buffer were boiled for 5 minutes and applied to 4‐12% NuPage Gels and separated by electrophoresis using the Xcell SureLock system (Invitrogen, Paisley, UK) under reducing conditions. Proteins were then transferred on to nitrocellulose membrane (Millipore, Bedford, UK) at 40 V overnight at 4°C using a Mini Protean II system (Bio‐Rad, Hemel Hempstead, UK). Following transfer, the PVDF was blocked (5% nonfat dry powdered milk, 0.1% Tween 20 in TBS) for 1 h and primary antibody was then added (1:1000 dilution) and incubated for 2 h at room temperature. Anti‐human GPVI was a kind gift from Dr. P. Smethurst, and anti‐β3 was obtained from Abcam, Cambridge, UK. Following washes with TBST, the membrane was incubated with HRP conjugated secondary antibody (1:10000 dilution/TBST) for 1 h at 24°C. The PVDF was developed using a chemiluminescent substrate (GE Healthcare, Amersham, Bucks, UK).

Lysates from freshly collected cortical tissue were prepared in RIPA buffer (Sigma), supplemented with protease and phosphatase inhibitors (Sigma), and homogenized using a Dounce homogenizer (Kimble Chase). Protein levels were quantified using BCA assays (Pierce Thermo Scientific). Proteins were resolved on NuPAGE gels (Invitrogen) using the XCell SureLock system (Invitrogen). The immunoreactive bands were detected by fluorescent ODYSSEY infrared imaging system (LI-COR). Equal protein loading was controlled by probing the blots with an antibody to β-actin. Antibodies used were rabbit anti-Smad1 (Abcam, 1:1000) and mouse anti-β-actin (Thermo Scientific, 1:2000).

One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was performed for comparative analysis of healthy and diseased samples on X Cell SureLock system (Invitrogen). Chemicals and reagents for 1D SDS-PAGE were purchased from Invitrogen (USA). β-mercaptoethanol, sucrose, and Tris HCL were purchased from Sigma Aldrich (USA).

For WB, freshly collected samples were lysed in RIPA buffer (Sigma) containing protease (Fisher Scientific) and phosphatase inhibitors (Sigma). Protein concentrations were determined using a BCA assay (Thermo Scientific). Proteins were resolved on 4–12% polyacrylamide NuPAGE gels (Invitrogen) using the XCell SureLock system (Invitrogen) and transferred to nitrocellulose membranes (Li-Cor Biosciences). The immunoreactive bands were detected by fluorescent ODYSSEY infrared imaging system (Li-Cor Biosciences). Equal protein loading was controlled by probing the blots with an antibody against a housekeeping control protein. Uncropped WBs can be found in the Source data file.

Fourth cohort of mice was used for Western blot analysis. Total protein fraction was extracted from hippocampus and olfactory bulbs. Samples were sonicated in ice-cold RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing 1× protease inhibitor (78430, Thermo Scientific) and centrifuged at 14,000× g for 10 min at 4 °C. Protein concentrations were determined with the BCA protein assay kit (23225, Thermo Scientific). Equal amounts of protein (20 μg) from each sample were separated on a NuPAGE Bis–Tris gel using the XCELL SureLock System (Invitrogen, USA). Thereafter, protein was transferred to a nitrocellulose membrane, after which the membranes were blocked and incubated with primary antibodies (Table 2) overnight at 4 °C. Immunoblots were then incubated with goat anti-rabbit (A11369, Invitrogen, USA) or goat anti-mouse (A-21057, Invitrogen, USA) fluorescent conjugated secondary antibodies for 1 h at room temperature, followed by visualization using a LI-COR Odyssey CLx system (LI-COR Biotechnologies, Lincoln, NE, USA). B-actin was used as a loading control.

Gel electrophoresis and western blotting were performed using the Xcell Surelock system (Invitrogen) using precast gradient gels (4–20%) as described [68] (link). The following antibodies were used: Anti-PDH-E1α (pSer293), Merck Millipore 1:10,000; and Anti-PDH-E1α, Abcam, 1:10,000. For visualisation of Western blots, HRP-based secondary antibodies were used followed by chemiluminescent detection on Kodak X-Omat film. Western blots were analysed by digitally scanning the blots, followed by densitometric analysis (ImageJ). For figure preparation of example western blots, linear adjustment of brightness/contrast was applied (Photoshop) equally across the image.