Laemli buffer

EO total lysates were prepared in Laemli buffer (BioRad) and beta-mercaptoethanol (BioRad) and then loaded in 10% SDS-polyacrylamide resolving gels (BioRad). The membranes were saturated with fat-free milk 5% in TBS (Tris-HCl 50 mM, NaCl 150 mM, pH 7.5) Tween 0.1% for 1 h at room temperature and probed with antibodies against ETC complexes (Total OXPHOS rodent WB Antibody Cocktail, Abcam) and beta-Actin (Santa Cruz). Signals were amplified and visualized with horseradish peroxidase-conjugated secondary antibodies (BioRad) and detected by the ChemiDoc^TM XRS + with Image Lab^TM Software (BioRad). Western blots images were analyzed using the ImageJ software (NIH).

For the detection of ApoA1, ApoB and APN in plasma and liver extracts from mice were resuspended in 40 µL of Laemli buffer (Bio-Rad) and heated at 95°C for 5 min. The samples were subjected to 10–20% linear gradient SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The presence of ApoA1, ApoB and APN was detected using anti-mouse ApoA1, ApoB and APN antibodies as primary and horseradish peroxidase-labeled rabbit anti-muse IgG as secondary antibody.

Plasmids containing the genes of interest were used to transform BL21ΔompA strain. Recombinant clones were grown in 200 ml LB medium (starting OD₆₀₀ = 0.05) and, when the cultures reached an OD₆₀₀ value of 0.5, protein expression was induced by addition of 1 mm IPTG. After 2 h, outer membrane vesicles (OMVs) were collected from culture supernatants by filtration through a 0.22 μm pore size filter (Millipore) followed by high-speed centrifugation (200,000 × g for 2 h). Pellets containing OMVs were finally resuspended in 1× PBS. Total bacterial lysates were prepared by suspending bacterial cells from 1 ml cultures (centrifuged at 13,000 × g for 5 min) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Laemli buffer (Bio-Rad, Hercules, CA) and heated at 100 °C for 5 min. Proteins were separated by 4–12% or 10% SDS-PAGE gel (Invitrogen, Carlsbad, CA), run in MES buffer (Invitrogen) and finally stained with Coomassie Blue.

All antibody details are listed in Supplementary Table S1. The procedure was carried out at RT unless otherwise noted. 1× TBS-T was used for all washing steps that were performed three times for 5 min after each antibody incubation. Briefly, rADSC and rADSC-EV pellets were resuspended in RIPA buffer, sonicated for 15 min and mixed with SDS-loading buffer (Laemli buffer, BioRad, Hercules, CA, USA). Subsequently, samples were denatured for 5 min at 99 °C, separated on a 12% SDS-PAGE and blotted onto PVDF membranes (BioRad). Membranes were blocked using 1× TBS-T with 5% BSA for 1.5 h and incubated with primary antibodies in 1× TBS–T with 5% BSA overnight at 4 °C. After washing, respective secondary antibodies were added for 1 h at RT. The blots were analyzed with the Odyssey^® CL × Imaging System (Li–Cor Biosciences, Lincoln, NE, USA) and Image Studio software (V5.2).

Purified viruses were treated using the PNGaseF kit (NEB) according to the manufacturer’s instructions. The samples were then treated with 4X-Laemli buffer (Bio-Rad) containing beta-mercaptoethanol (BME) and heated at 100 °C for 5 min. Approximately 1 ug purified virus was added to each lane on a 7.5% Mini-PROTEAN® TGX™ precast protein gel (Bio-Rad) and run for 5.5 h at 25 V. After electrophoresis, the proteins were transferred to a nitrocellulose membrane using the iBlot transfer and stack device (Invitrogen) at 20 V for 7 min. The membranes were blocked in 5% nonfat dry milk (Bio-Rad) in PBS-T (0.5%) for 1 h at room temperature with shaking. The blocking solution was removed and anti-N2 guinea pig antisera (generated in-house by vaccinating guinea pigs intramuscularly with Hong Kong14 N2 recombinant protein) diluted 1:2000 in PBS supplemented with 1% (w/v) BSA was added. The membranes were incubated overnight and washed three times with PBS-T. The following day, a donkey anti-guinea pig IgG-horseradish peroxidase conjugate (IgG-HRP; EMD Millipore, AP193P) was added for 1 h at room temperature and the membranes were washed. The membranes were developed by adding ECL prime and incubated for 5 min. The developed blot membranes were visualized by scanning using an ImageScanner III imager with accompanying LabScan software (GE Healthcare).

Mice were sacrificed and the cortex and thalamus were harvested from half cerebrums. The tissues were homogenized in ice-cold RIPA buffer (1% NP-40, 0.5% C₂₄H₃₉Na0₄, 0.1%SDS, 2 mM EDTA) dissolved in 1xPBS along with a protease inhibitor cocktail (Roche) and centrifuged for 20 min at 4°C. Protein concentration was measured with bicinchoninic acid (BCA) assay (Smith et al., 1985 (link)) and 100 μg were mixed with 4x laemli buffer (Biorad). Tissue lysates were loaded in 12% SDS-PAGE [ddH₂0, 30% acrylamide-bis (29:1), 1.5M Tris-CL pH 8.8, 10%SDS, 10%APS, Temed] and transferred to a Hybond PVDF blotting membrane (Life Sciences) using a semi-dry unit. The membrane was blocked in blocking solution [5% non-fat skimmed milk in PBS containing 0.1% Tween-20 (PBS-T)] for 1 h at room temperature and then incubated overnight at 4°C with the following mouse primary antibodies: anti-Cx43 (Millipore, 1:1,000) and anti-Tubulin-E7 (DSHB, 1:4,000) and the rabbit anti-Cx30 (Thermo Fisher Scientific, 1:500). After 15 min washes in PBS-T, membranes were incubated for 1 h at room temperature with a goat anti-mouse or anti-rabbit HRP-conjugated secondary antibody (Jackson Immunoresearch, 1:3,000). The bound antibody was visualized by enhanced chemiluminescence system (ECL, GE Healthcare Life Sciences), protein expression was normalized against tubulin and band intensity was quantified using Image J.