Turboblot

Whole-cell lysates for immunoblotting were prepared by dissolving cells in Laemmli Sample Buffer containing 5% 2-mercaptoethanol (Bio-Rad). Protein samples were loaded on 4–15% polyacrylamide gels (BioRad) and transferred onto 0.45 µm pore-size nitrocellulose membranes (Bio-Rad) using the Turboblot (BioRad). After that, blots were blocked with 5% milk for 1 h at room temperature. For phospho-tau, 5% BSA in TBS was used for blocking. Blots were incubated overnight at 4 °C with primary antibodies. And then blots were probed with secondary antibodies for 1 h at room temperature before ECL development and imaging (Bio-Rad). The primary antibodies used for immunoblotting are as follows: Lamin A/C antibody, (Abcam, 1:750); Lamin B1 antibody (Santa Cruz, 1:200); APP antibody (BioLegend, 1:400), total tau antibody (Santa Cruz, 1:200), PHF6 p-tau antibody (Santa Cruz, 1:200), γ-H2AX antibody (Abcam, 1:3000), and β-actin (1:5000, Sigma-Aldrich).

Total protein lysate was quantified using a standard Bradford assay and 10 μg of lysate was used for immunoblotting experiments. For Blue Native PAGE, cells were lysed in 1x Sample Preparation buffer (ThermoFisher) containing 1% digitonin. 1% SDS was supplemented for SDS-PAGE. All proteins were transferred to PVDF membranes using TurboBlot (Bio-rad) at 2.5 mA for seven mins. The primary antibodies used were anti-FLAG (M2, Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GAPDH (Santa Cruz Biotechnology Cat# sc-47724, RRID:AB_627678) and anti-HA (Y-11, Santa Cruz Biotechnology Cat# sc-805, RRID:AB_631618). The secondary antibodies were anti-rabbit IgG-HRP (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567), anti-mouse IgG-HRP (light chain specific) (Jackson ImmunoResearch Labs Cat# 205-032-176, RRID:AB_2339056) and anti-mouse IgG-HRP (Jackson ImmunoResearch Labs Cat# 115-035-003, RRID:AB_10015289).

Cells were lysed in RIPA buffer, supplemented with 1% SDS, protease (Sigma P8340) and phosphatase inhibitors (Roche 04906845001). Lysates were separated on 12%, 15% or 4–20% SDS-PAGE and transferred onto PVDF membrane (Bio-Rad TurboBlot). Membranes were blocked in 5% BSA in TBS-T (0.05%) and incubated overnight with primary antibodies. Blots were washed, probed with the appropriate secondary antibodies and processed with ECL (film or Bio-Rad chemiluminescent system) or imaged on the SpectraMax i3 platform with ScanLater module (Molecular Devices).

Equal volume of PE/PF from fraction four of each fractioned sample was mixed to form protein pools of study and control groups, respectively. After SDS-PAGE electrophoresis, proteins were transferred to a nitrocellulose membrane from an SDS-PAGE gel using a semidry transfer apparatus following the instructions provided by the manufacturer (TurboBlot, Bio-Rad, USA). The membrane was then probed with an anti-omentin-1 monoclonal antibody (Clontech, USA) using the chemiluminescent detection system (GE Healthcare, USA). The images were recorded with VersaDoc MP4000 (Bio-Rad, USA) and a set of prestained protein markers (Fermentas, USA) was used to assess the size of the signal (~40 kD) generated in western blots. For the purpose of spot analysis, ImageJ, freely available software, was used. The integrated density of each protein band was measured by outlining them and using the analyze/measure command.

For protein analysis, cells or tissue cryosections were lysed in RIPA buffer (50 mM TRIS, 150 mM, NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, protease inhibitor, ddH₂O) and 25 μg of protein was electrophorezed in 10% precast NuPAGE gels (Invitrogen), 1 h at 180 V. The proteins were transferred to nitrocellulose membranes (Hybond-C Extra, Amersham, Freiburg, Germany) by semidry blotting (Turbo Blot BioRad, Munich, Germany) at 20 V, 25 min. Membranes were blocked for 1 h at room temperature (RT) in TBS (50 mM Tris, 150 mM NaCl, pH 7.5, 5% fat-free dry milk and 2.5% casein) and washed in TBST (0.05% Tween 20 in PBS), 2 x 5 min at RT. As primary antibody rabbit anti-claudin-3 antibody (1:3000, Acris, Herford, Germany), rabbit anti-claudin-4 antibody (1:3000, Acris), rabbit anti-CPE (1:4000, Acris), mouse monoclonal anti-β-tubulin (1:1000, BD Bioscience, MD, USA) or mouse monoclonal anti-β-actin antibody (1:10000, Sigma-Aldrich, MI, USA) was added respectively over night at 4 °C and washed in TBST. As secondary HRP-labeled goat anti-rabbit-IgG antibody (1:10000, Promega, Madison, WI, USA) or goat anti-mouse IgM/IgG (1:10000, Sigma-Aldrich) was added for 1 h, RT. Membranes were washed in TBST. Detection was done using ECL solution (Amersham) and exposure to Kodak X-Omat AR film (Kodak, Stuttgart, Germany).

pUC19 plasmid was compacted by TFAM as above and split into acetylation reactions in which mock-incubated pUC19 plasmid (plasmid DNA in the incubation buffer), TFAM alone, 50 bp:TFAM, or 25 bp:TFAM samples were incubated with 5 mM lithium-acetyl-CoA (Sigma) for 1.5 h or 3 h. Each sample was split to maintain an equal amount of TFAM per sample. After incubation, each acetylation reaction was split for Western blot, EMSA, and SDS-PAGE analyses. For SDS-PAGE analysis, a 4 μl aliquot of 50 bp:TFAM and 2 μl aliquots of the TFAM only and 25 bp:TFAM samples (23 ng TFAM) were boiled and run on a 12.5% SDS-PAGE. For EMSA analysis, 9 μl of each reaction (92 ng TFAM) was loaded onto a 1% agarose gel with 10% glycerol and run at 4 °C for ∼3.5 h in the EMSA running buffer listed before. For Western blot analysis, 16 μl of 50 bp:TFAM reaction and 8 μl of each 25 bp:TFAM reaction and TFAM alone (190 ng TFAM) was boiled and run on 12.5% SDS-PAGE. Protein was transferred onto a nitrocellulose membrane (BioRad) using a Turboblot (BioRad). Detection used anti–acetyl-lysine antibody (Cell Signaling Antibody) according to manufacturer’s details and goat-anti-rabbit horseradish peroxidase-linked secondary antibodies. Image analysis was conducted using ImageQuant software (see SI).

A quantity of 50–250 μg of protein sample were loaded on 8–15% polyacrylamide gels and transferred onto PVDF membrane (Merck Millipore) or Amersham Hybond‐ECL nitrocellulose membranes (GE Healthcare) using the Turboblot (BioRad) or a wet transfer system (BioRad). Blots were incubated overnight at 4°C with primary antibodies, probed with secondary antibodies and developed using ECL (Perkin Elmer). The following primary antibodies were used: anti‐BAX (1:1,000, Cell Signaling #2772), anti‐actin (1:10,000, Cell Signaling #4967), anti‐GAPDH (1:5,000, Santa Cruz sc‐47724), anti‐PARP (1:1,000, Cell Signaling #9542), anti‐BAK (1:1,000, Cell Signaling, #3814), anti‐BID (1:1,000, 2002S Cell Signaling), anti‐BOK (1:1,000, Abcam 186745), anti‐α‐tubulin (1:5,000 #2144 Cell Signaling), anti‐VDAC (1:1,000, D73D12, Cell Signaling), anti‐Smac/Diablo (1:1,000, D5S3R, Cell Signaling), anti‐cytochrome c (1:1,000, BD Pharmingen 556433) and anti‐GFP from mouse IgG1κ (1:1,000, clones 7.1 and 13.1, Roche).