Xt reducing agent

dECM extracts were prepared from CMSC29 and DMSC23. Protein concentrations of each sample were measured using the Pierce protein assay (Thermo Scientific) according to the manufacturer’s protocol with bovine serum albumin as the standard. The protein extracts were resuspended in 7.5 μl XT Sample Buffer supplemented with 1.5 μl XT reducing agent (Bio-Rad), heated to 95°C for 10 mins, and centrifuged at 14,000 g for 2 mins. After centrifugation, each sample was loaded into a 1.0 mm 4–12% gradient Bis-Tris Criterion XT Precast Gel (Bio-Rad) along with Precision Plus protein standard (Bio-Rad). Fibronectin (Life Technologies) and Collagen I (Trevigen, MD, USA) were loaded as high molecular weight protein controls. SDS-PAGE was performed at 150V for 100 mins using XT MOPS buffer (Bio-Rad). The gel was stained with Coomassie Brilliant Blue G-250 overnight and destained in deionized H₂O for 1 hour. Visualization of the bands was carried out on a densitometer (GE ImageScanner III) and the image was taken using ImageQuant TL software (GE Healthcare).

Ketamine/xylazine was purchased from Sigma-Aldrich. ¹⁴C-sucrose and optiphase supermix scintillation cocktail were purchased from PerkinElmer. ³H-Sumatriptan was purchased from American Radiolabeled Chemicals Inc. Sumatriptan was generously donated by the Porreca Lab (University of Arizona; source: Abmole Bioscience). Topiramate was purchased from Cayman Chemical. TS-2 tissue solubilizer was purchased from Research Products International. EDTA-free complete protease inhibitors were purchased from Roche. The Coomassie Plus Better Bradford Assay kit was purchased from Thermo Scientific. XT sample buffer, XT reducing agent, Precision Plus dual color prestained molecular weight markers, and TGX criterion gels were purchased from Bio-Rad. Primary antibodies used for Western blotting (WB) include the following: claudin-5 [Invitrogen (Thermo Fisher), catalog #4C3C2, 1:500], occludin [Invitrogen (Thermo Fisher), catalog #OC-3F10, 1:1000], and α-tubulin (Cell Signaling, catalog #DM1A, 1:5000). Secondary antibodies for WB were purchased from Cell Signaling Technology and used at a 1:20,000 dilution. All other chemicals were purchased from Sigma-Aldrich.

To demonstrate Ag expression by the AdHu5-OVA and MVA-OVA vectors, we detected OVA expression by Western blotting of the supernatants of infected cell cultures. HEK293A cells were cultured in RPMI 1640 medium with 10% FCS and infected with either AdHu5-OVA or AdHu5 expressing the irrelevant malaria Ag PfAMA1 (36 (link)) at a multiplicity of infection of 10 infectious units/cell. BHK cells were cultured similarly and infected with either MVA-OVA or a MVA-PfAMA1 (36 (link)) at a multiplicity of infection of 10 PFU/cell. Cell supernatant and infected cells were collected separately 8 and 24 h postinfection, boiled with XT reducing agent and Laemlli buffer (both from Bio-Rad), run on a Novex NuPAGE Bis-Tris SDS 4–12% gel (ThermoFisher), transferred to nitrocellulose membrane using a TransBlot Turbo system (Bio-Rad), and blotted using an iBind system (ThermoFisher). Serum from mice immunized with OVA protein was used as primary Ab, with detection using alkaline-phosphatase–conjugated donkey-anti-mouse IgG (The Jackson Laboratory) and BCIP/NBT (Sigma-Aldrich). Recombinant OVA (InvivoGen) was used as a positive control.

Cells were collected, washed twice with 1× PBS and homogenated in RIPA buffer freshly supplemented with proteases inhibitors. To detect COQ7 protein, 60 μg of proteins from the sample extracts was electrophoresed in 12% Mini-PROTEAN TGX^™ precast gels (Bio-Rad) using the electrophoresis system mini-PROTEAN Tetra Cell (Bio-Rad). To detect COQ9 protein, 60 μg of proteins from sample extracts was prepared in XT sample buffer + XT-reducing agent (Bio-Rad) and electrophoresed in a 10% Criterion^™ XT precast gel (Bio-Rad) using MOPS running buffer and the electrophoresis system Criterion Cell (Bio-Rad). In all experiments, proteins were transferred onto PVDF 0.45 μm membranes using a mini Trans-blot Cell (Bio-rad) or Trans-blot Cell (Bio-Rad) and probed with target antibodies. Protein-antibody interactions were detected with peroxidase-conjugated horse anti-mouse, anti-rabbit or anti-goat IgG antibodies using Amersham ECL^™ Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK). Band quantification was carried out using an Image Station 2000R (Kodak, Spain) and a Kodak 1D 3.6 software. COQ7 and COQ9 protein band intensity was normalized to VDAC1, and the data expressed in terms of percent relative to wild-type mice [4 (link)].

For SDS-PAGE analysis, the pellets containing the cytoplasmic contents were treated with CelLytic™ B Cell Lysis Reagent (Sigma-Aldrich). An additional 50 units/mL benzonase nuclease (Sigma-Aldrich) was used for all samples. The samples were incubated for 30 min at room temperature with shaking (100 rpm). Dilutions (5x) of the samples were done with XT MES Running Buffer (Bio-Rad). Each of the samples was combined with XT Sample Buffer 2X (Bio-Rad) and with XT Reducing Agent (Bio-Rad) and incubated at 95 °C for 5 min. Samples (10 μL, 10x diluted) and a ladder (Precision Plus Protein™ Dual Color Standards, Bio-Rad, 5 μL) were loaded on SDS-gel (Criterion™ XT Bis-Tris Precast Gels, 12%, Bio-Rad) for gel electrophoresis (200 V, 45 min), which after end of run was stained with InstantBlue™ Coomassie Protein Stain (Expedeon).

SDS-PAGE was used to compare the compositions of the dECMs. Protein extracts were prepared using the Pierce Protein Assay Kit according to manufacturer instructions and resuspended in 7.5 μL XT sample buffer/1.5 μL XT reducing agent (Bio-Rad), placed in a 95°C heating block for 10 min, and centrifuged for 2 min at 14,000 g. Samples were loaded into 1.0 mm 4–12% gradient Bis-Tris Criterion XT Precast Gel (Bio-Rad), with collagen I (Sigma Aldrich) and Precision Plus protein ladder (Bio-Rad) used as controls. Loaded gels were placed into XT MOPS buffer (Bio-Rad) and electrophoresed at 150 V for ~100 min. Gels were then stained with BioSafe Coomassie (Bio-Rad) for at least 2 h and washed with deionized water three times for 5 min each. Bands were visualized using a GE Image Scanner III, with images taken using ImageQuant TL software (GE Healthcare).