Tgx criterion gels

For MS analysis, EV-containing density fractions (1.06–1.13 g/ml) were diluted in PBS containing 0.1% BSA and centrifuged at 100,000×g for 90 min in an SW32 rotor. EV pellets were resuspended in reducing Laemmli sample buffer and denatured at 100 °C for 4 min. The protein content of EV preparations was estimated by SDS-PAGE followed by SYPRO^® Ruby protein staining densitometry. In short, sample aliquots were used for protein separation on 8–16% TGX-Criterion gels (Bio-Rad, Hercules, CA). After electrophoresis, gels were fixed in 40% methanol/10% acetic acid and stained with SYPRO^® Ruby (Invitrogen, Carlsbad, CA), followed by destaining in 10% methanol/6% acetic acid. Gels were imaged on a Bio-Rad ChemiDoc imaging system and densitometry quantitation was performed using Image Lab software (Bio-Rad). For LC-MS/MS analysis, EV-protein input was equalized between conditions and proteins were separated by SDS-PAGE on 8–16% TGX-Criterion gels. Gels were fixed in 50% methanol/10% acetic acid, stained with Coomassie Brilliant Blue R-250 (Bio-Rad), and destained sequentially with 40% methanol/10% acetic acid and Milli-Q. In-gel digestion using trypsin (Promega, Madison, WI) was performed as previously described^{78 (link)}. After extraction with 100% acetonitrile the samples were dried and reconstituted in 10% formic acid/5% DMSO.

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.

Immunoblotting was performed as previously described 14 (link) with the following modifications. Briefly, cell lysates were obtained in SDS lysis buffer consisting of 2% SDS, 50 mM Tris-HCl (pH 6.8), 100 mM DTT, 5% Ficoll 400, 0.0001% bromophenol blue and a cocktail of protease inhibitors. Lysates were sonicated on ice before heating at 97^oC for 7 min to completely denature the protein. Protein concentration was determined using IR spectroscopy (which is insensitive to high SDS and DTT concentrations) with a Millipore DirectDetect spectrometer. Lysates were equally loaded at 10-15 µg per lane on BioRad TGX Criterion gels (4-15%) and resolved at 300 V in Tris-Glycine running buffer. Proteins were transferred on nitrocellulose membranes using the BioRad TransBlot turbo according to the manufacturer's instructions. Membranes were blocked in fish serum (Aqua Block) or 5% nonfat dry milk in TBS-T (0.05% Tween 20) and probed overnight at 4^oC with primary antibodies as indicated in Supplementary Table S1. The membranes were washed in TBS-T and incubated with secondary antibodies for 1 h at room temperature. After washing, semi-quantitative visualization of the proteins of interest was enabled using an enhanced chemiluminescence reagent as previously described 15 (link) and the Azure C400 western blot imaging system.